Human cytokine receptor

ABSTRACT

Cytokines and their receptors have proven usefulness in both basic research and as therapeutics. The present invention provides a new human cytokine receptor designated as “Zcytor 18.”

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application is a continuation of U.S. patentapplication Ser. No. 09/912,157, filed Jul. 24, 2001, which claims thebenefit of U.S. Provisional Application Serial No. 60/220,747, filedJul. 26, 2000, both of which are herein incorporated by reference.

TECHNICAL FIELD

[0002] The present invention relates generally to a new proteinexpressed by human cells. In particular, the present invention relatesto a novel gene that encodes a receptor, designated as “Zcytor 18,” andto nucleic acid molecules encoding Zcytor 18 polypeptides.

BACKGROUND OF THE INVENTION

[0003] Cytokines are soluble, small proteins that mediate a variety ofbiological effects, including the regulation of the growth anddifferentiation of many cell types (see, for example, Arai et al., Annu.Rev. Biochem. 59:783 (1990); Mosmann, Curr. Opin. Immunol. 3:311 (1991);Paul and Seder, Cell 76:241 (1994)). Proteins that constitute thecytokine group include interleukins, interferons, colony stimulatingfactors, tumor necrosis factors, and other regulatory molecules. Forexample, human interleukin-17 is a cytokine which stimulates theexpression of interleukin-6, intracellular adhesion molecule 1,interleukin-8, granulocyte macrophage colony-stimulating factor, andprostaglandin E2 expression, and plays a role in the preferentialmaturation of CD34+hematopoietic precursors into neutrophils (Yao etal., J. Immunol. 155:5483 (1995); Fossiez et al., J. Exp. Med. 183:2593(1996)).

[0004] Receptors that bind cytokines are typically composed of one ormore integral membrane proteins that bind the cytokine with highaffinity and transduce this binding event to the cell through thecytoplasmic portions of the certain receptor subunits. Cytokinereceptors have been grouped into several classes on the basis ofsimilarities in their extracellular ligand binding domains. For example,the receptor chains responsible for binding and/or transducing theeffect of interferons are members of the type II cytokine receptorfamily, based upon a characteristic 200 residue extracellular domain.

[0005] The demonstrated in vivo activities of cytokines and theirreceptors illustrate the clinical potential of, and need for, othercytokines, cytokine receptors, cytokine agonists, and cytokineantagonists.

BRIEF SUMMARY OF THE INVENTION

[0006] The present invention provides a novel receptor, designated“Zcytor 18.” The present invention also provides Zcytor 18 polypeptidesand Zcytor 18 fusion proteins, as well as nucleic acid moleculesencoding such polypeptides and proteins, and methods for using thesenucleic acid molecules and amino acid sequences.

DETAILED DESCRIPTION OF THE INVENTION

[0007] 1. Overview

[0008] An illustrative nucleotide sequence that encodes Zcytor 18 isprovided by The encoded polypeptide has the following amino acidsequence: MAPWLQLCSV FFTVNACLNG SQLAVAAGGS GRARGADTCG (SEQ ID NO:2)WRMKAAARPR LCVANEGVGP ASRNSGLYNI TFKYDNCTTY LNPVGKHVIA DAQNITISQYACHIDQVAVTI LWSPGALGLE FLKGFRVILE ELKSEGRQCQ QLILKDPKQL NSSFKRTGMESQPFLNMKFE TDYFVKVVPF PSIKNESNYH PFFFRTRACD LLLQPDNLAC KPFWKPRNLNISQHGSDMQV SFDHAPHNEG FRFFYLHYKL KHEGPFKRKT CKQEQTTETT SCLLQNVSPGDYIIELVDDT NTTRKVMHYA LKPVHSPWAG PIIRAVA1TVP LVVISAFATL FTVMCRKKQQENIYSHLDEE SSESSTYTAA LPRERLRPRP KVFLCYSSKD GQNHMNVVQC FAYFLQDFCGCEVALDLWED FSLCREGQRE WVIQKIHESQ FIIVVCSKGM KYFVDKKNYK HKGGGRGSGKGELFLVAVSA IAEKLRQAKQ SSSAALSKFI AVYFDYSCEG DVPGIILDLST KYRLMDNLPQLCSHLHSRDH GLQEPGQHTR QGSRRNYFRS KSGRSLYVAI CNMHQFIIDEE PDWFEKQFVPFHPPPLRYRE PVLEKFDSGL VLNDVMCKPG PESDFCLKVE AAVLGATGPA DSQHESQHGGLDQDGEARPA LDGSAALQPL LHTVKAGSPS DMPRDSGLYD SSVPSSELSL PLMEGLSTDQTETSSLTESV SSSSGLGEEE PPALPSKLLS SGSCKADLGC RSYTDELHAV APL.

[0009] Features of the Zcytor 18 polypeptide include an extracellulardomain at amino acid residues 1 to 313 of SEQ ID NO:2, a putative signalsequence at amino acid residues 1 to 35 of SEQ ID NO:2, a transmembranedomain at amino acid residues 314 to 335 of SEQ ID NO:2, and anintracellular domain at amino acid residues 336 to 753 of SEQ ID NO:2.The Zcytor 18 gene resides in human chromosome 3p14.3.

[0010] Northern analysis revealed that the Zcytor 18 gene is stronglyexpressed in testicular, ovarian, and uterine tissue, and moderatelyexpressed in fetal heart, fetal kidney, fetal skin, and adult brain. Incontrast, little expression was detected in muscle, bladder, adultkidney, adult lung, fetal small intestine, salivary gland, or adrenalgland, and expression was not detectable in spleen, thymus, peripheralblood leukocytes, pancreas, liver, placenta, thyroid, lymph node, orbone marrow. Studies also indicate that Zcytor 18 gene expression ishigher in breast tissue than in normal breast tissue. Thus, Zcytor 18nucleotide and amino acid sequences can be used to differentiatetissues.

[0011] One variant form of Zcytor 18 is characterized by the followingamino acid substitutions in the amino acid sequence of SEQ ID NO:2:Thr²⁶⁹ to Met²⁶⁹, and Val⁷⁵⁰ to Ala⁷⁵⁰. Additional variants of humanZcytor 18 can be identified by comparison with the murine sequence (SEQID NO:12), such as Leu²⁴⁶ to Val²⁴⁶, and Lys²⁵⁷ to Arg²⁵⁷. Nucleotide,amino acid, and degenerate sequences of the human variant form areprovided as SEQ ID NOs:4, 5, and 6, respectively.

[0012] A splice variant of Zcytor 18 lacks amino acid residues 43 to 56of SEQ ID NO:2. Nucleotide, amino acid, and degenerate sequences of thevariant form are provided as SEQ ID NOs:7, 8, and 9, respectively.Features of this Zcytor 18 splice variant include an extracellulardomain at amino acid residues 1 to 299 of SEQ ID NO:8, a putative signalsequence at amino acid residues 1 to 35 of SEQ ID NO:8, a transmembranedomain at amino acid residues 300 to 321 of SEQ ID NO:8, and anintracellular domain at amino acid residues 322 to 739 of SEQ ID NO:8.

[0013] As described below, the present invention provides isolatedpolypeptides comprising an amino acid sequence that is at least 70%, atleast 80%, or at least 90% identical to a reference amino acid sequenceselected from the group consisting of: (a) an amino acid sequencecomprising amino acid residues 36 to 189 of SEQ ID NO:2, (b) amino acidresidues 1 to 189 of SEQ ID NO:2, (c) amino acid residues 36 to 313 ofSEQ ID NO:2, (d) amino acid residues 336 to 753 of SEQ ID NO:2, (e)amino acid residues 36 to 753 of SEQ ID NO:2, (f) amino acid residues 1to 753 of SEQ ID NO:2, (g) amino acid resides 1 to 299 of SEQ ID NO:8,(h) amino acid residues 36 to 299 of SEQ ID NO:8, (i) amino acidresidues 36 to 175 of SEQ ID NO:8, (j) amino acid residues 1 to 300 ofSEQ ID NO:12, (k) amino acid residues 36 to 300 of SEQ ID NO:12, and (l)1 to 739 of SEQ ID NO:12. Certain of these polypeptides can specificallybind with an antibody that specifically binds with a polypeptideconsisting of the amino acid sequence of SEQ ID NO:2, SEQ ID NO:8, orSEQ ID NO:12. Illustrative polypeptides include polypeptides comprising,or consisting of, amino acid residues 36 to 189 of SEQ ID NO:2, aminoacid residues 36 to 753 of SEQ ID NO:2, 36 to 299 of SEQ ID NO:8, aminoacid resides 36 to 175 of SEQ ID NO:8, or amino acid residues 36 to 300of SEQ ID NO:12.

[0014] The present invention also provides isolated polypeptidescomprising at least 15 contiguous amino acid residues of an amino acidsequence selected from the group consisting of: (a) amino acid residues1 to 203 of SEQ ID NO:2, (b) amino acid residues 36 to 203 of SEQ IDNO:2, (c) amino acid residues 36 to 313 of SEQ ID NO:2, (d) amino acidresidues 1 to 753 of SEQ ID NO:2, (e) amino acid residues 1 to 189 ofSEQ ID NO:8, (f) amino acid residues 36 to 189 of SEQ ID NO:8, (g) aminoacid residues 36 to 299 of SEQ ID NO:8, (h) amino acid residues 1 to 739of SEQ ID NO:8, (i) amino acid residues 1 to 300 of SEQ ID NO:12, (j)amino acid residues 36 to 300 of SEQ ID NO:12, and (k) 1 to 739 of SEQID NO:12. The present invention further provides isolated polypeptidescomprising at least 30 contiguous amino acid residues of an amino acidsequence selected from the group consisting of: (l) amino acid residues1 to 218 of SEQ ID NO:2, (m) amino acid residues 36 to 218 of SEQ IDNO:2, (n) amino acid residues 36 to 313 of SEQ ID NO:2, (o) amino acidresidues 1 to 753 of SEQ ID NO:2, (p) amino acid residues 1 to 204 ofSEQ ID NO:8, (q) amino acid residues 36 to 204 of SEQ ID NO:8, (r) aminoacid residues 36 to 299 of SEQ ID NO:8, (s) amino acid residues 1 to 739of SEQ ID NO:8, (t) amino acid residues 1 to 300 of SEQ ID NO:12, (u)amino acid residues 36 to 300 of SEQ ID NO:12, and (v) 1 to 739 of SEQID NO:12. Illustrative polypeptides include polypeptides that eithercomprise, or consist of, amino acid residues (a) to (v).

[0015] The present invention also includes variant Zcytor 18polypeptides, wherein the amino acid sequence of the variant polypeptideshares an identity with amino acid residues 36 to 189 of SEQ ID NO:2,amino acid residues 36 to 175 of SEQ ID NO:8, or amino acid residues 36to 300 of SEQ ID NO:12, selected from the group consisting of at least70% identity, at least 80% identity, at least 90% identity, at least 95%identity, or greater than 95% identity, and wherein any differencebetween the amino acid sequence of the variant polypeptide and thecorresponding amino acid sequence of SEQ ID NO:2, SEQ ID NO:8, or SEQ IDNO:12, is due to one or more conservative amino acid substitutions. Inaddition, the present invention includes variant Zcytor 18 polypeptides,characterized by the following amino acid substitutions in the aminoacid sequence of SEQ ID NO:2: Thr²⁶⁹ to Met²⁶⁹, and Val⁷⁵⁰ to Ala⁷⁵⁰.

[0016] The polypeptides described herein can further comprise anaffinity tag.

[0017] The present invention further provides antibodies and antibodyfragments that specifically bind with such polypeptides. Exemplaryantibodies include polyclonal antibodies, murine monoclonal antibodies,humanized antibodies derived from murine monoclonal antibodies, andhuman monoclonal antibodies. Illustrative antibody fragments includeF(ab′)₂, F(ab)₂, Fab′, Fab, Fv, scFv, and minimal recognition units. Thepresent invention further includes compositions comprising a carrier anda peptide, polypeptide, or antibody described herein.

[0018] The present invention also provides isolated nucleic acidmolecules that encode a Zcytor 18 polypeptide, wherein the nucleic acidmolecule is selected from the group consisting of: (a) a nucleic acidmolecule comprising the nucleotide sequence of SEQ ID NO:3 or thenucleotide sequence of SEQ ID NO:9, (b) a nucleic acid molecule encodingan amino acid sequence that comprises amino acid residues 36 to 189 ofSEQ ID NO:2, amino acid residues 36 to 313 of SEQ ID NO:2, amino acidresidues 36 to 175 of SEQ ID NO:8, or amino acid residues 36 to 299 ofSEQ ID NO:8, and (c) a nucleic acid molecule that remains hybridizedfollowing stringent wash conditions to a nucleic acid moleculeconsisting of the nucleotide sequence of nucleotides 192 to 652 of SEQID NO:1, the nucleotide sequence of nucleotides 192 to 610 of SEQ IDNO:7; the complement of the nucleotide sequence of nucleotides 192 to652 of SEQ ID NO:1; or the complement of the nucleotide sequence ofnucleotides 192 to 610 of SEQ ID NO:7. Illustrative nucleic acidmolecules include those in which any difference between the amino acidsequence encoded by nucleic acid molecule (c) and the correspondingamino acid sequence of SEQ ID NO:2, or SEQ ID NO:8, is due to aconservative amino acid substitution. The present invention furthercontemplates isolated nucleic acid molecules that comprise nucleotides192 to 652 of SEQ ID NO:1 or nucleotides 192 to 610 of SEQ ID NO:7, aswell as nucleic acid molecules that comprise nucleotides 206 to 1000 ofSEQ ID NO:11.

[0019] The present invention also includes vectors and expressionvectors comprising such nucleic acid molecules. Such expression vectorsmay comprise a transcription promoter, and a transcription terminator,wherein the promoter is operably linked with the nucleic acid molecule,and wherein the nucleic acid molecule is operably linked with thetranscription terminator. The present invention further includesrecombinant host cells and recombinant viruses comprising these vectorsand expression vectors. Illustrative host cells include avian,bacterial, yeast, fungal, insect, mammalian, and plant cells.Recombinant host cells comprising such expression vectors can be used toproduce Zcytor 18 polypeptides by culturing such recombinant host cellsthat comprise the expression vector and that produce the Zcytor 18protein, and, optionally, isolating the Zcytor 18 protein from thecultured recombinant host cells. The present invention includes theprotein products of such processes.

[0020] In addition, the present invention provides pharmaceuticalcompositions comprising a pharmaceutically acceptable carrier and atleast one of such an expression vector or recombinant virus comprisingsuch expression vectors. The present invention further includespharmaceutical compositions, comprising a pharmaceutically acceptablecarrier and a polypeptide described herein.

[0021] The present invention also contemplates methods for detecting thepresence of Zcytor 18 RNA in a biological sample, comprising the stepsof (a) contacting a Zcytor 18 nucleic acid probe under hybridizingconditions with either (i) test RNA molecules isolated from thebiological sample, or (ii) nucleic acid molecules synthesized from theisolated RNA molecules, wherein the probe has a nucleotide sequencecomprising a portion of the nucleotide sequence of SEQ ID NO:1, or itscomplement, and (b) detecting the formation of hybrids of the nucleicacid probe and either the test RNA molecules or the synthesized nucleicacid molecules, wherein the presence of the hybrids indicates thepresence of Zcytor 18 RNA in the biological sample. For example,suitable probes consist of the following nucleotide sequences:nucleotides 86 to 652 of SEQ ID NO:1, nucleotides 192 to 652 of SEQ IDNO:1, nucleotides 86 to 1024 of SEQ ID NO:1, nucleotides 192 to 1024 ofSEQ ID NO:1, nucleotides 86 to 610 of SEQ ID NO:7, nucleotides 192 to610 of SEQ ID NO:7, nucleotides 86 to 982 of SEQ ID NO:7, andnucleotides 192 to 982 of SEQ ID NO:7. Other suitable probes consist ofthe complement of these nucleotide sequences, or a portion of thenucleotide sequences or their complements. An example of a biologicalsample is a human biological sample, such as a biopsy or autopsyspecimen.

[0022] The present invention further provides methods for detecting thepresence of Zcytor 18 polypeptide in a biological sample, comprising thesteps of: (a) contacting the biological sample with an antibody or anantibody fragment that specifically binds with a polypeptide consistingof the amino acid sequence of SEQ ID NO:2, or SEQ ID NO:8, wherein thecontacting is performed under conditions that allow the binding of theantibody or antibody fragment to the biological sample, and (b)detecting any of the bound antibody or bound antibody fragment. Such anantibody or antibody fragment may further comprise a detectable labelselected from the group consisting of radioisotope, fluorescent label,chemiluminescent label, enzyme label, bioluminescent label, andcolloidal gold. An example of a biological sample is a human biologicalsample, such as a biopsy or autopsy specimen.

[0023] The present invention also provides kits for performing thesedetection methods. For example, a kit for detection of Zcytor 18 geneexpression may comprise a container that comprises a nucleic acidmolecule, wherein the nucleic acid molecule is selected from the groupconsisting of (a) a nucleic acid molecule comprising the nucleotidesequence of nucleotides 86 to 652 of SEQ ID NO:1, (b) a nucleic acidmolecule comprising the complement of nucleotides 192 to 652 of thenucleotide sequence of SEQ ID NO:1, (c) a nucleic acid moleculecomprising the nucleotide sequence of nucleotides 86 to 610 of SEQ IDNO:7, (d) a nucleic acid molecule comprising the complement ofnucleotides 192 to 610 of the nucleotide sequence of SEQ ID NO:7, and(e) a nucleic acid molecule that is a fragment of (a)-(d) consisting ofat least eight nucleotides. Such a kit may also comprise a secondcontainer that comprises one or more reagents capable of indicating thepresence of the nucleic acid molecule. On the other hand, a kit fordetection of Zcytor 18 protein may comprise a container that comprisesan antibody, or an antibody fragment, that specifically binds with apolypeptide consisting of the amino acid sequence of SEQ ID NO:2 or SEQID NO:8.

[0024] The present invention also contemplates anti-idiotype antibodies,or anti-idiotype antibody fragments, that specifically bind an antibodyor antibody fragment that specifically binds a polypeptide consisting ofthe amino acid sequence of SEQ ID NO:2 or SEQ ID NO:8. An exemplaryanti-idiotype antibody binds with an antibody that specifically binds apolypeptide consisting of amino acid residues 36 to 313 of SEQ ID NO:2,amino acid residues 36 to 189 of SEQ ID NO:2, amino acid residues 36 to299 of SEQ ID NO:8, or amino acid residues 36 to 175 of SEQ ID NO:8.

[0025] The present invention also provides isolated nucleic acidmolecules comprising a nucleotide sequence that encodes a Zcytor 18secretion signal sequence and a nucleotide sequence that encodes abiologically active polypeptide, wherein the Zcytor 18 secretion signalsequence comprises an amino acid sequence of residues 1 to 35 of SEQ IDNO:2. Illustrative biologically active polypeptides include Factor VIIa,proinsulin, insulin, follicle stimulating hormone, tissue typeplasminogen activator, tumor necrosis factor, interleukin, colonystimulating factor, interferon, erythropoietin, and thrombopoietin.Moreover, the present invention provides fusion proteins comprising aZcytor 18 secretion signal sequence and a polypeptide, wherein theZcytor 18 secretion signal sequence comprises an amino acid sequence ofresidues 1 to 35 of SEQ ID NO:2.

[0026] The present invention also provides fusion proteins, comprising aZcytor 18 polypeptide and an immunoglobulin moiety. In such fusionproteins, the immunoglobulin moiety may be an immunoglobulin heavy chainconstant region, such as a human Fc fragment. The present inventionfurther includes isolated nucleic acid molecules that encode such fusionproteins.

[0027] These and other aspects of the invention will become evident uponreference to the following detailed description. In addition, variousreferences are identified below and are incorporated by reference intheir entirety.

[0028] 2. Definitions

[0029] In the description that follows, a number of terms are usedextensively. The following definitions are provided to facilitateunderstanding of the invention.

[0030] As used herein, “nucleic acid” or “nucleic acid molecule” refersto polynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleicacid (RNA), oligonucleotides, fragments generated by the polymerasechain reaction (PCR), and fragments generated by any of ligation,scission, endonuclease action, and exonuclease action. Nucleic acidmolecules can be composed of monomers that are naturally-occurringnucleotides (such as DNA and RNA), or analogs of naturally-occurringnucleotides (e.g., α-enantiomeric forms of naturally-occurringnucleotides), or a combination of both. Modified nucleotides can havealterations in sugar moieties and/or in pyrimidine or purine basemoieties. Sugar modifications include, for example, replacement of oneor more hydroxyl groups with halogens, alkyl groups, amines, and azidogroups, or sugars can be functionalized as ethers or esters. Moreover,the entire sugar moiety can be replaced with sterically andelectronically similar structures, such as aza-sugars and carbocyclicsugar analogs. Examples of modifications in a base moiety includealkylated purines and pyrimidines, acylated purines or pyrimidines, orother well-known heterocyclic substitutes. Nucleic acid monomers can belinked by phosphodiester bonds or analogs of such linkages. Analogs ofphosphodiester linkages include phosphorothioate, phosphorodithioate,phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate,phosphoranilidate, phosphoramidate, and the like. The term “nucleic acidmolecule” also includes so-called “peptide nucleic acids,” whichcomprise naturally-occurring or modified nucleic acid bases attached toa polyamide backbone. Nucleic acids can be either single stranded ordouble stranded.

[0031] The term “complement of a nucleic acid molecule” refers to anucleic acid molecule having a complementary nucleotide sequence andreverse orientation as compared to a reference nucleotide sequence. Forexample, the sequence 5′ ATGCACGGG 3′ is complementary to 5′ CCCGTGCAT3′.

[0032] The term “contig” denotes a nucleic acid molecule that has acontiguous stretch of identical or complementary sequence to anothernucleic acid molecule. Contiguous sequences are said to “overlap” agiven stretch of a nucleic acid molecule either in their entirety oralong a partial stretch of the nucleic acid molecule.

[0033] The term “degenerate nucleotide sequence” denotes a sequence ofnucleotides that includes one or more degenerate codons as compared to areference nucleic acid molecule that encodes a polypeptide. Degeneratecodons contain different triplets of nucleotides, but encode the sameamino acid residue (i.e., GAU and GAC triplets each encode Asp).

[0034] The term “structural gene” refers to a nucleic acid molecule thatis transcribed into messenger RNA (mRNA), which is then translated intoa sequence of amino acids characteristic of a specific polypeptide.

[0035] An “isolated nucleic acid molecule” is a nucleic acid moleculethat is not integrated in the genomic DNA of an organism. For example, aDNA molecule that encodes a growth factor that has been separated fromthe genomic DNA of a cell is an isolated DNA molecule. Another exampleof an isolated nucleic acid molecule is a chemically-synthesized nucleicacid molecule that is not integrated in the genome of an organism. Anucleic acid molecule that has been isolated from a particular speciesis smaller than the complete DNA molecule of a chromosome from thatspecies.

[0036] A “nucleic acid molecule construct” is a nucleic acid molecule,either single- or double-stranded, that has been modified through humanintervention to contain segments of nucleic acid combined and juxtaposedin an arrangement not existing in nature.

[0037] “Linear DNA” denotes non-circular DNA molecules having free 5′and 3′ ends. Linear DNA can be prepared from closed circular DNAmolecules, such as plasmids, by enzymatic digestion or physicaldisruption.

[0038] “Complementary DNA (cDNA)” is a single-stranded DNA molecule thatis formed from an mRNA template by the enzyme reverse transcriptase.Typically, a primer complementary to portions of mRNA is employed forthe initiation of reverse transcription. Those skilled in the art alsouse the term “cDNA” to refer to a double-stranded DNA moleculeconsisting of such a single-stranded DNA molecule and its complementaryDNA strand. The term “cDNA” also refers to a clone of a cDNA moleculesynthesized from an RNA template.

[0039] A “promoter” is a nucleotide sequence that directs thetranscription of a structural gene. Typically, a promoter is located inthe 5′ non-coding region of a gene, proximal to the transcriptionalstart site of a structural gene. Sequence elements within promoters thatfunction in the initiation of transcription are often characterized byconsensus nucleotide sequences. These promoter elements include RNApolymerase binding sites, TATA sequences, CAAT sequences,differentiation-specific elements (DSEs; McGehee et al., Mol.Endocrinol. 7:551(1993)), cyclic AMP response elements (CREs), serumresponse elements (SREs; Treisman, Seminars in Cancer Biol. 1:47(1990)), glucocorticoid response elements (GREs), and binding sites forother transcription factors, such as CRE/ATF (O'Reilly et al., J. Biol.Chem. 267:19938 (1992)), AP2 (Ye et al., J. Biol. Chem. 269:25728(1994)), SPI, cAMP response element binding protein (CREB; Loeken, GeneExpr. 3:253 (1993)) and octamer factors (see, in general, Watson et al.,eds., Molecular Biology of the Gene, 4th ed. (The Benjamin/CummingsPublishing Company, Inc. 1987), and Lemaigre and Rousseau, Biochem. J.303:1 (1994)). If a promoter is an inducible promoter, then the rate oftranscription increases in response to an inducing agent. In contrast,the rate of transcription is not regulated by an inducing agent if thepromoter is a constitutive promoter. Repressible promoters are alsoknown.

[0040] A “core promoter” contains essential nucleotide sequences forpromoter function, including the TATA box and start of transcription. Bythis definition, a core promoter may or may not have detectable activityin the absence of specific sequences that may enhance the activity orconfer tissue specific activity.

[0041] A “regulatory element” is a nucleotide sequence that modulatesthe activity of a core promoter. For example, a regulatory element maycontain a nucleotide sequence that binds with cellular factors enablingtranscription exclusively or preferentially in particular cells,tissues, or organelles. These types of regulatory elements are normallyassociated with genes that are expressed in a “cell-specific,”“tissue-specific,” or “organelle-specific” manner.

[0042] An “enhancer” is a type of regulatory element that can increasethe efficiency of transcription, regardless of the distance ororientation of the enhancer relative to the start site of transcription.

[0043] “Heterologous DNA” refers to a DNA molecule, or a population ofDNA molecules, that does not exist naturally within a given host cell.DNA molecules heterologous to a particular host cell may contain DNAderived from the host cell species (i.e., endogenous DNA) so long asthat host DNA is combined with non-host DNA (i.e., exogenous DNA). Forexample, a DNA molecule containing a non-host DNA segment encoding apolypeptide operably linked to a host DNA segment comprising atranscription promoter is considered to be a heterologous DNA molecule.Conversely, a heterologous DNA molecule can comprise an endogenous geneoperably linked with an exogenous promoter. As another illustration, aDNA molecule comprising a gene derived from a wild-type cell isconsidered to be heterologous DNA if that DNA molecule is introducedinto a mutant cell that lacks the wild-type gene.

[0044] A “polypeptide” is a polymer of amino acid residues joined bypeptide bonds, whether produced naturally or synthetically. Polypeptidesof less than about 10 amino acid residues are commonly referred to as“peptides.”

[0045] A “protein” is a macromolecule comprising one or more polypeptidechains. A protein may also comprise non-peptidic components, such ascarbohydrate groups. Carbohydrates and other non-peptidic substituentsmay be added to a protein by the cell in which the protein is produced,and will vary with the type of cell. Proteins are defined herein interms of their amino acid backbone structures; substituents such ascarbohydrate groups are generally not specified, but may be presentnonetheless.

[0046] A peptide or polypeptide encoded by a non-host DNA molecule is a“heterologous” peptide or polypeptide.

[0047] An “integrated genetic element” is a segment of DNA that has beenincorporated into a chromosome of a host cell after that element isintroduced into the cell through human manipulation. Within the presentinvention, integrated genetic elements are most commonly derived fromlinearized plasmids that are introduced into the cells byelectroporation or other techniques. Integrated genetic elements arepassed from the original host cell to its progeny.

[0048] A “cloning vector” is a nucleic acid molecule, such as a plasmid,cosmid, or bacteriophage, which has the capability of replicatingautonomously in a host cell. Cloning vectors typically contain one or asmall number of restriction endonuclease recognition sites that allowinsertion of a nucleic acid molecule in a determinable fashion withoutloss of an essential biological function of the vector, as well asnucleotide sequences encoding a marker gene that is suitable for use inthe identification and selection of cells transformed with the cloningvector. Marker genes typically include genes that provide tetracyclineresistance or ampicillin resistance.

[0049] An “expression vector” is a nucleic acid molecule encoding a genethat is expressed in a host cell. Typically, an expression vectorcomprises a transcription promoter, a gene, and a transcriptionterminator. Gene expression is usually placed under the control of apromoter, and such a gene is said to be “operably linked to” thepromoter. Similarly, a regulatory element and a core promoter areoperably linked if the regulatory element modulates the activity of thecore promoter.

[0050] A “recombinant host” is a cell that contains a heterologousnucleic acid molecule, such as a cloning vector or expression vector. Inthe present context, an example of a recombinant host is a cell thatproduces Zcytor 18 from an expression vector. In contrast, Zcytor 18 canbe produced by a cell that is a “natural source” of Zcytor 18, and thatlacks an expression vector.

[0051] “Integrative transformants” are recombinant host cells, in whichheterologous DNA has become integrated into the genomic DNA of thecells.

[0052] A “fusion protein” is a hybrid protein expressed by a nucleicacid molecule comprising nucleotide sequences of at least two genes. Forexample, a fusion protein can comprise at least part of a Zcytor 18polypeptide fused with a polypeptide that binds an affinity matrix. Sucha fusion protein provides a means to isolate large quantities of Zcytor18 using affinity chromatography.

[0053] The term “receptor” denotes a cell-associated protein that bindsto a bioactive molecule termed a “ligand.” This interaction mediates theeffect of the ligand on the cell. Receptors can be membrane bound,cytosolic or nuclear; monomeric (e.g., thyroid stimulating hormonereceptor, beta-adrenergic receptor) or multimeric (e.g., PDGF receptor,growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSF receptor,erythropoietin receptor and IL-6 receptor). Membrane-bound receptors arecharacterized by a multi-domain structure comprising an extracellularligand-binding domain and an intracellular effector domain that istypically involved in signal transduction. In certain membrane-boundreceptors, the extracellular ligand-binding domain and the intracellulareffector domain are located in separate polypeptides that comprise thecomplete functional receptor.

[0054] In general, the binding of ligand to receptor results in aconformational change in the receptor that causes an interaction betweenthe effector domain and other molecule(s) in the cell, which in turnleads to an alteration in the metabolism of the cell. Metabolic eventsthat are often linked to receptor-ligand interactions include genetranscription, phosphorylation, dephosphorylation, increases in cyclicAMP production, mobilization of cellular calcium, mobilization ofmembrane lipids, cell adhesion, hydrolysis of inositol lipids andhydrolysis of phospholipids.

[0055] The term “secretory signal sequence” denotes a DNA sequence thatencodes a peptide (a “secretory peptide”) that, as a component of alarger polypeptide, directs the larger polypeptide through a secretorypathway of a cell in which it is synthesized. The larger polypeptide iscommonly cleaved to remove the secretory peptide during transit throughthe secretory pathway.

[0056] An “isolated polypeptide” is a polypeptide that is essentiallyfree from contaminating cellular components, such as carbohydrate,lipid, or other proteinaceous impurities associated with the polypeptidein nature. Typically, a preparation of isolated polypeptide contains thepolypeptide in a highly purified form, i.e., at least about 80% pure, atleast about 90% pure, at least about 95% pure, greater than 95% pure, orgreater than 99% pure. One way to show that a particular proteinpreparation contains an isolated polypeptide is by the appearance of asingle band following sodium dodecyl sulfate (SDS)-polyacrylamide gelelectrophoresis of the protein preparation and Coomassie Brilliant Bluestaining of the gel. However, the term “isolated” does not exclude thepresence of the same polypeptide in alternative physical forms, such asdimers or alternatively glycosylated or derivatized forms.

[0057] The terms “amino-terminal” and “carboxyl-terminal” are usedherein to denote positions within polypeptides. Where the contextallows, these terms are used with reference to a particular sequence orportion of a polypeptide to denote proximity or relative position. Forexample, a certain sequence positioned carboxyl-terminal to a referencesequence within a polypeptide is located proximal to the carboxylterminus of the reference sequence, but is not necessarily at thecarboxyl terminus of the complete polypeptide.

[0058] The term “expression” refers to the biosynthesis of a geneproduct. For example, in the case of a structural gene, expressioninvolves transcription of the structural gene into mRNA and thetranslation of mRNA into one or more polypeptides.

[0059] The term “splice variant” is used herein to denote alternativeforms of RNA transcribed from a gene. Splice variation arises naturallythrough use of alternative splicing sites within a transcribed RNAmolecule, or less commonly between separately transcribed RNA molecules,and may result in several mRNAs transcribed from the same gene. Splicevariants may encode polypeptides having altered amino acid sequence. Theterm splice variant is also used herein to denote a polypeptide encodedby a splice variant of an mRNA transcribed from a gene.

[0060] As used herein, the term “immunomodulator” includes cytokines,stem cell growth factors, lymphotoxins, co-stimulatory molecules,hematopoietic factors, and synthetic analogs of these molecules.

[0061] The term “complement/anti-complement pair” denotes non-identicalmoieties that form a non-covalently associated, stable pair underappropriate conditions. For instance, biotin and avidin (orstreptavidin) are prototypical members of a complement/anti-complementpair. Other exemplary complement/anti-complement pairs includereceptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs,sense/antisense polynucleotide pairs, and the like. Where subsequentdissociation of the complement/anti-complement pair is desirable, thecomplement/anti-complement pair preferably has a binding affinity ofless than 10⁹ M⁻¹.

[0062] An “anti-idiotype antibody” is an antibody that binds with thevariable region domain of an immunoglobulin. In the present context, ananti-idiotype antibody binds with the variable region of an anti-Zcytor18 antibody, and thus, an anti-idiotype antibody mimics an epitope ofZcytor 18.

[0063] An “antibody fragment” is a portion of an antibody such asF(ab′)₂, F(ab)₂, Fab′, Fab, and the like. Regardless of structure, anantibody fragment binds with the same antigen that is recognized by theintact antibody. For example, an anti-Zcytor 18 monoclonal antibodyfragment binds with an epitope of Zcytor 18.

[0064] The term “antibody fragment” also includes a synthetic or agenetically engineered polypeptide that binds to a specific antigen,such as polypeptides consisting of the light chain variable region, “Fv”fragments consisting of the variable regions of the heavy and lightchains, recombinant single chain polypeptide molecules in which lightand heavy variable regions are connected by a peptide linker (“scFvproteins”), and minimal recognition units consisting of the amino acidresidues that mimic the hypervariable region.

[0065] A “chimeric antibody” is a recombinant protein that contains thevariable domains and complementary determining regions derived from arodent antibody, while the remainder of the antibody molecule is derivedfrom a human antibody.

[0066] “Humanized antibodies” are recombinant proteins in which murinecomplementarity determining regions of a monoclonal antibody have beentransferred from heavy and light variable chains of the murineimmunoglobulin into a human variable domain.

[0067] As used herein, a “therapeutic agent” is a molecule or atom,which is conjugated to an antibody moiety to produce a conjugate, whichis useful for therapy. Examples of therapeutic agents include drugs,toxins, immunomodulators, chelators, boron compounds, photoactive agentsor dyes, and radioisotopes.

[0068] A “detectable label” is a molecule or atom, which can beconjugated to an antibody moiety to produce a molecule useful fordiagnosis. Examples of detectable labels include chelators, photoactiveagents, radioisotopes, fluorescent agents, paramagnetic ions, or othermarker moieties.

[0069] The term “affinity tag” is used herein to denote a polypeptidesegment that can be attached to a second polypeptide to provide forpurification or detection of the second polypeptide or provide sites forattachment of the second polypeptide to a substrate. In principal, anypeptide or protein for which an antibody or other specific binding agentis available can be used as an affinity tag. Affinity tags include apolyhistidine tract, protein A (Nilsson et al., EMBO J. 4:1075 (1985);Nilsson et al., Methods Enzymol. 198:3 (1991)), glutathione Stransferase (Smith and Johnson, Gene 67:31 (1988)), Glu-Glu affinity tag(Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952 (1985)),substance P, FLAG peptide (Hopp et al., Biotechnology 6:1204 (1988)),streptavidin binding peptide, or other antigenic epitope or bindingdomain. See, in general, Ford et al., Protein Expression andPurification 2:95 (1991). DNA molecules encoding affinity tags areavailable from commercial suppliers (e.g., Pharmacia Biotech,Piscataway, N.J.).

[0070] A “naked antibody” is an entire antibody, as opposed to anantibody fragment, which is not conjugated with a therapeutic agent.Naked antibodies include both polyclonal and monoclonal antibodies, aswell as certain recombinant antibodies, such as chimeric and humanizedantibodies.

[0071] As used herein, the term “antibody component” includes both anentire antibody and an antibody fragment.

[0072] An “immunoconjugate” is a conjugate of an antibody component witha therapeutic agent or a detectable label.

[0073] As used herein, the term “antibody fusion protein” refers to arecombinant molecule that comprises an antibody component and a Zcytor18 polypeptide component. Examples of an antibody fusion protein includea protein that comprises a Zcytor 18 extracellular domain, and either anFc domain or an antigen-biding region.

[0074] A “target polypeptide” or a “target peptide” is an amino acidsequence that comprises at least one epitope, and that is expressed on atarget cell, such as a tumor cell, or a cell that carries an infectiousagent antigen. T cells recognize peptide epitopes presented by a majorhistocompatibility complex molecule to a target polypeptide or targetpeptide and typically lyse the target cell or recruit other immune cellsto the site of the target cell, thereby killing the target cell.

[0075] An “antigenic peptide” is a peptide, which will bind a majorhistocompatibility complex molecule to form an MHC-peptide complex,which is recognized by a T cell, thereby inducing a cytotoxic lymphocyteresponse upon presentation to the T cell. Thus, antigenic peptides arecapable of binding to an appropriate major histocompatibility complexmolecule and inducing a cytotoxic T cells response, such as cell lysisor specific cytokine release against the target cell, which binds orexpresses the antigen. The antigenic peptide can be bound in the contextof a class I or class II major histocompatibility complex molecule, onan antigen presenting cell or on a target cell.

[0076] In eukaryotes, RNA polymerase II catalyzes the transcription of astructural gene to produce mRNA. A nucleic acid molecule can be designedto contain an RNA polymerase II template in which the RNA transcript hasa sequence that is complementary to that of a specific mRNA. The RNAtranscript is termed an “anti-sense RNA” and a nucleic acid moleculethat encodes the anti-sense RNA is termed an “anti-sense gene.”Anti-sense RNA molecules are capable of binding to mRNA molecules,resulting in an inhibition of mRNA translation.

[0077] An “anti-sense oligonucleotide specific for Zcytor 18” or a“Zcytor 18 anti-sense oligonucleotide” is an oligonucleotide having asequence (a) capable of forming a stable triplex with a portion of theZcytor 18 gene, or (b) capable of forming a stable duplex with a portionof an mRNA transcript of the Zcytor 18 gene.

[0078] A “ribozyme” is a nucleic acid molecule that contains a catalyticcenter. The term includes RNA enzymes, self-splicing RNAs, self-cleavingRNAs, and nucleic acid molecules that perform these catalytic functions.A nucleic acid molecule that encodes a ribozyme is termed a “ribozymegene.”

[0079] An “external guide sequence” is a nucleic acid molecule thatdirects the endogenous ribozyme, RNase P, to a particular species ofintracellular mRNA, resulting in the cleavage of the mRNA by RNase P. Anucleic acid molecule that encodes an external guide sequence is termedan “external guide sequence gene.”

[0080] The term “variant Zcytor 18 gene” refers to nucleic acidmolecules that encode a polypeptide having an amino acid sequence thatis a modification of SEQ ID NO:2. Such variants includenaturally-occurring polymorphisms of Zcytor 18 genes, as well assynthetic genes that contain conservative amino acid substitutions ofthe amino acid sequence of SEQ ID NO:2. Additional variant forms ofZcytor 18 genes are nucleic acid molecules that contain insertions ordeletions of the nucleotide sequences described herein. A variant Zcytor18 gene can be identified, for example, by determining whether the genehybridizes with a nucleic acid molecule having the nucleotide sequenceof SEQ ID NO:1, or its complement, under stringent conditions.

[0081] Alternatively, variant Zcytor 18 genes can be identified bysequence comparison. Two amino acid sequences have “100% amino acidsequence identity” if the amino acid residues of the two amino acidsequences are the same when aligned for maximal correspondence.Similarly, two nucleotide sequences have “100% nucleotide sequenceidentity” if the nucleotide residues of the two nucleotide sequences arethe same when aligned for maximal correspondence. Sequence comparisonscan be performed using standard software programs such as those includedin the LASERGENE bioinformatics computing suite, which is produced byDNASTAR (Madison, Wis.). Other methods for comparing two nucleotide oramino acid sequences by determining optimal alignment are well-known tothose of skill in the art (see, for example, Peruski and Peruski, TheInternet and the New Biology: Tools for Genomic and Molecular Research(ASM Press, Inc. 1997), Wu et al. (eds.), “Information Superhighway andComputer Databases of Nucleic Acids and Proteins,” in Methods in GeneBiotechnology, pages 123-151 (CRC Press, Inc. 1997), and Bishop (ed.),Guide to Human Genome Computing, 2nd Edition (Academic Press, Inc.1998)). Particular methods for determining sequence identity aredescribed below.

[0082] Regardless of the particular method used to identify a variantZcytor 18 gene or variant Zcytor 18 polypeptide, a variant gene orpolypeptide encoded by a variant gene may be functionally characterizedthe ability to bind specifically to an anti-Zcytor 18 antibody.

[0083] The term “allelic variant” is used herein to denote any of two ormore alternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inphenotypic polymorphism within populations. Gene mutations can be silent(no change in the encoded polypeptide) or may encode polypeptides havingaltered amino acid sequence. The term allelic variant is also usedherein to denote a protein encoded by an allelic variant of a gene.

[0084] The term “ortholog” denotes a polypeptide or protein obtainedfrom one species that is the functional counterpart of a polypeptide orprotein from a different species. Sequence differences among orthologsare the result of speciation. “Paralogs” are distinct but structurallyrelated proteins made by an organism. Paralogs are believed to arisethrough gene duplication. For example, α-globin, β-globin, and myoglobinare paralogs of each other.

[0085] The present invention includes functional fragments of Zcytor 18genes. Within the context of this invention, a “functional fragment” ofa Zcytor 18 gene refers to a nucleic acid molecule that encodes aportion of a Zcytor 18 polypeptide, which is a domain described hereinor at least specifically binds with an anti-Zcytor 18 antibody.

[0086] Due to the imprecision of standard analytical methods, molecularweights and lengths of polymers are understood to be approximate values.When such a value is expressed as “about” X or “approximately” X, thestated value of X will be understood to be accurate to ±10%.

[0087] 3. Production of Nucleic Acid Molecules Encoding Zcytor 18

[0088] Nucleic acid molecules encoding a human Zcytor 18 can be obtainedby screening a human cDNA or genomic library using polynucleotide probesbased upon SEQ ID NO:1. These techniques are standard andwell-established.

[0089] As an illustration, a nucleic acid molecule that encodes a humanZcytor 18 can be isolated from a cDNA library. In this case, the firststep would be to prepare the cDNA library by isolating RNA from atissue, such as testicular tissue, using methods well-known to those ofskill in the art. In general, RNA isolation techniques must provide amethod for breaking cells, a means of inhibiting RNase-directeddegradation of RNA, and a method of separating RNA from DNA, protein,and polysaccharide contaminants. For example, total RNA can be isolatedby freezing tissue in liquid nitrogen, grinding the frozen tissue with amortar and pestle to lyse the cells, extracting the ground tissue with asolution of phenol/chloroform to remove proteins, and separating RNAfrom the remaining impurities by selective precipitation with lithiumchloride (see, for example, Ausubel et al. (eds.), Short Protocols inMolecular Biology, 3d Edition, pages 4-1 to 4-6 (John Wiley & Sons 1995)[“Ausubel (1995)”]; Wu et al., Methods in Gene Biotechnology, pages33-41 (CRC Press, Inc. 1997) [“Wu (1997)”]).

[0090] Alternatively, total RNA can be isolated by extracting groundtissue with guanidinium isothiocyanate, extracting with organicsolvents, and separating RNA from contaminants using differentialcentrifugation (see, for example, Chirgwin et al., Biochemistry 18:52(1979); Ausubel (1995) at pages 4-1 to 4-6; Wu (1997) at pages 3341).

[0091] In order to construct a cDNA library, poly(A)+ RNA must beisolated from a total RNA preparation. Poly(A)+ RNA can be isolated fromtotal RNA using the standard technique of oligo(dT)-cellulosechromatography (see, for example, Aviv and Leder, Proc. Nat'l Acad. Sci.USA 69:1408 (1972); Ausubel (1995) at pages 4-11 to 412).

[0092] Double-stranded cDNA molecules are synthesized from poly(A)⁺ RNAusing techniques well-known to those in the art. (see, for example, Wu(1997) at pages 41-46). Moreover, commercially available kits can beused to synthesize double-stranded cDNA molecules. For example, suchkits are available from Life Technologies, Inc. (Gaithersburg, Md.),CLONTECH Laboratories, Inc. (Palo Alto, Calif.), Promega Corporation(Madison, Wis.) and STRATAGENE (La Jolla, Calif.).

[0093] Various cloning vectors are appropriate for the construction of acDNA library. For example, a cDNA library can be prepared in a vectorderived from bacteriophage, such as a λgt10 vector. See, for example,Huynh et al., “Constructing and Screening cDNA Libraries in λgt10 andλgt11,” in DNA Cloning: A Practical Approach Vol. I, Glover (ed.), page49 (IRL Press, 1985); Wu (1997) at pages 47-52.

[0094] Alternatively, double-stranded cDNA molecules can be insertedinto a plasmid vector, such as a PBLUESCRIPT vector (STRATAGENE; LaJolla, Calif.), a LAMDAGEM-4 (Promega Corp.) or other commerciallyavailable vectors. Suitable cloning vectors also can be obtained fromthe American Type Culture Collection (Manassas, Va.).

[0095] To amplify the cloned cDNA molecules, the cDNA library isinserted into a prokaryotic host, using standard techniques. Forexample, a cDNA library can be introduced into competent E. coli DH5cells, which can be obtained, for example, from Life Technologies, Inc.(Gaithersburg, Md.).

[0096] A human genomic library can be prepared by means well-known inthe art (see, for example, Ausubel (1995) at pages 5-1 to 5-6; Wu (1997)at pages 307-327). Genomic DNA can be isolated by lysing tissue with thedetergent Sarkosyl, digesting the lysate with proteinase K, clearinginsoluble debris from the lysate by centrifugation, precipitatingnucleic acid from the lysate using isopropanol, and purifyingresuspended DNA on a cesium chloride density gradient.

[0097] DNA fragments that are suitable for the production of a genomiclibrary can be obtained by the random shearing of genomic DNA or by thepartial digestion of genomic DNA with restriction endonucleases. GenomicDNA fragments can be inserted into a vector, such as a bacteriophage orcosmid vector, in accordance with conventional techniques, such as theuse of restriction enzyme digestion to provide appropriate termini, theuse of alkaline phosphatase treatment to avoid undesirable joining ofDNA molecules, and ligation with appropriate ligases. Techniques forsuch manipulation are well-known in the art (see, for example, Ausubel(1995) at pages 5-1 to 5-6; Wu (1997) at pages 307327).

[0098] Alternatively, human genomic libraries can be obtained fromcommercial sources such as Research Genetics (Huntsville, Ala.) and theAmerican Type Culture Collection (Manassas, Va.).

[0099] A library containing cDNA or genomic clones can be screened withone or more polynucleotide probes based upon SEQ ID NO:1, using standardmethods (see, for example, Ausubel (1995) at pages 6-1 to 6-11).

[0100] Nucleic acid molecules that encode a human Zcytor 18 gene canalso be obtained using the polymerase chain reaction (PCR) witholigonucleotide primers having nucleotide sequences that are based uponthe nucleotide sequences of the Zcytor 18 gene, as described herein.General methods for screening libraries with PCR are provided by, forexample, Yu et al., “Use of the Polymerase Chain Reaction to ScreenPhage Libraries,” in Methods in Molecular Biology, Vol. 15: PCRProtocols: Current Methods and Applications, White (ed.), pages 211-215(Humana Press, Inc. 1993). Moreover, techniques for using PCR to isolaterelated genes are described by, for example, Preston, “Use of DegenerateOligonucleotide Primers and the Polymerase Chain Reaction to Clone GeneFamily Members,” in Methods in Molecular Biology, Vol. 15: PCRProtocols: Current Methods and Applications, White (ed.), pages 317337(Humana Press, Inc. 1993).

[0101] Anti-Zcytor 18 antibodies, produced as described below, can alsobe used to isolate DNA sequences that encode human Zcytor 18 genes fromcDNA libraries. For example, the antibodies can be used to screen λgt11expression libraries, or the antibodies can be used for immunoscreeningfollowing hybrid selection and translation (see, for example, Ausubel(1995) at pages 6-12 to 6-16; Margolis et al., “Screening λ expressionlibraries with antibody and protein probes,” in DNA Cloning 2:Expression Systems, 2nd Edition, Glover et al. (eds.), pages 1-14(Oxford University Press 1995)).

[0102] As an alternative, a Zcytor 18 gene can be obtained bysynthesizing nucleic acid molecules using mutually priming longoligonucleotides and the nucleotide sequences described herein (see, forexample, Ausubel (1995) at pages 8-8 to 8-9). Established techniquesusing the polymerase chain reaction provide the ability to synthesizeDNA molecules at least two kilobases in length (Adang et al., PlantMolec. Biol. 21:1131 (1993), Bambot et al., PCR Methods and Applications2:266 (1993), Dillon et al., “Use of the Polymerase Chain Reaction forthe Rapid Construction of Synthetic Genes,” in Methods in MolecularBiology, Vol. 15: PCR Protocols: Current Methods and Applications, White(ed.), pages 263-268, (Humana Press, Inc. 1993), and Holowachuk et al.,PCR Methods Appl. 4:299 (1995)).

[0103] The nucleic acid molecules of the present invention can also besynthesized with “gene machines” using protocols such as thephosphoramidite method. If chemically-synthesized double stranded DNA isrequired for an application such as the synthesis of a gene or a genefragment, then each complementary strand is made separately. Theproduction of short genes (60 to 80 base pairs) is technicallystraightforward and can be accomplished by synthesizing thecomplementary strands and then annealing them. For the production oflonger genes (>300 base pairs), however, special strategies may berequired, because the coupling efficiency of each cycle during chemicalDNA synthesis is seldom 100%. To overcome this problem, synthetic genes(double-stranded) are assembled in modular form from single-strandedfragments that are from 20 to 100 nucleotides in length. For reviews onpolynucleotide synthesis, see, for example, Glick and Pasternak,Molecular Biotechnology, Principles and Applications of Recombinant DNA(ASM Press 1994), Itakura et al., Annu. Rev. Biochem. 53:323 (1984), andClimie et al., Proc. Nat'l Acad. Sci. USA 87:633 (1990).

[0104] The sequence of a Zcytor 18 cDNA or Zcytor 18 genomic fragmentcan be determined using standard methods. Zcytor 18 polynucleotidesequences disclosed herein can also be used as probes or primers toclone 5′ non-coding regions of a Zcytor 18 gene. Promoter elements froma Zcytor 18 gene can be used to direct the expression of heterologousgenes in, for example, transgenic animals or patients treated with genetherapy. The identification of genomic fragments containing a Zcytor 18promoter or regulatory element can be achieved using well-establishedtechniques, such as deletion analysis (see, generally, Ausubel (1995)).

[0105] Cloning of 5′ flanking sequences also facilitates production ofZcytor 18 proteins by “gene activation,” as disclosed in U.S. Pat. No.5,641,670. Briefly, expression of an endogenous Zcytor 18 gene in a cellis altered by introducing into the Zcytor 18 locus a DNA constructcomprising at least a targeting sequence, a regulatory sequence, anexon, and an unpaired splice donor site. The targeting sequence is aZcytor 18 5′ non-coding sequence that permits homologous recombinationof the construct with the endogenous Zcytor 18 locus, whereby thesequences within the construct become operably linked with theendogenous Zcytor 18 coding sequence. In this way, an endogenous Zcytor18 promoter can be replaced or supplemented with other regulatorysequences to provide enhanced, tissue-specific, or otherwise regulatedexpression.

[0106] 4. Production of Zcytor 18 Variants

[0107] The present invention provides a variety of nucleic acidmolecules, including DNA and RNA molecules, which encode the Zcytor 18polypeptides disclosed herein. Those skilled in the art will readilyrecognize that, in view of the degeneracy of the genetic code,considerable sequence variation is possible among these polynucleotidemolecules. SEQ ID NO:3 is a degenerate nucleotide sequence thatencompasses all nucleic acid molecules that encode the Zcytor 18polypeptide of SEQ ID NO:2. Those skilled in the art will recognize thatthe degenerate sequence of SEQ ID NO:3 also provides all RNA sequencesencoding SEQ ID NO:2, by substituting U for T. Thus, the presentinvention contemplates Zcytor 18 polypeptide-encoding nucleic acidmolecules comprising nucleotide 86 to nucleotide 2344 of SEQ ID NO:1,and their RNA equivalents. Similarly, the present invention contemplatesZcytor 18 polypeptide-encoding nucleic acid molecules comprisingnucleotide 86 to nucleotide 2302 of SEQ ID NO:7, and their RNAequivalents.

[0108] Table 1 sets forth the one-letter codes used within SEQ ID NO:3to denote degenerate nucleotide positions. “Resolutions” are thenucleotides denoted by a code letter. “Complement” indicates the codefor the complementary nucleotide(s). For example, the code Y denoteseither C or T, and its complement R denotes A or G, A beingcomplementary to T, and G being complementary to C. TABLE 1 NucleotideResolution Complement Resolution A A T T C C G G G G C C T T A A R A|G YC|T Y C|T R A|G M A|C K G|T K G|T M A|C S C|G S C|G W A|T W A|T H A|C|TD A|G|T B C|G|T V A|C|G V A|C|G B C|G|T D A|G|T H A|C|T N A|C|G|T NA|C|G|T

[0109] The degenerate codons used in SEQ ID NO:3, encompassing allpossible codons for a given amino acid, are set forth in Table 2. TABLE2 One Amino Letter Degenerate Acid Code Codons Codon Cys C TGC TGT TGYSer S AGC AGT TCA TCC TCG TCT WSN Thr T ACA ACC ACG ACT ACN Pro P CCACCC CCG CCT CCN Ala A GCA GCC GCG GCT GCN Gly G GGA GGC GGG GGT GGN AsnN AAC AAT AAY Asp D GAC GAT GAY Glu E GAA GAG GAR Gln Q CAA CAG CAR HisH CAC CAT CAY Arg R AGA AGG CGA CGC CGG CGT MGN Lys K AAA AAG AAR Met MATG ATG Ile I ATA ATC ATT ATH Leu L CTA CTC CTG CTT TTA TTG YTN Val VGTA GTC GTG GTT GTN Phe F TTC TTT TTY Tyr Y TAC TAT TAY Trp W TGG TGGTer . TAA TAG TGA TRR Asn|Asp B RAY Glu|Gln Z SAR Any X NNN

[0110] One of ordinary skill in the art will appreciate that someambiguity is introduced in determining a degenerate codon,representative of all possible codons encoding an amino acid. Forexample, the degenerate codon for serine (WSN) can, in somecircumstances, encode arginine (AGR), and the degenerate codon forarginine (MGN) can, in some circumstances, encode serine (AGY). Asimilar relationship exists between codons encoding phenylalanine andleucine. Thus, some polynucleotides encompassed by the degeneratesequence may encode variant amino acid sequences, but one of ordinaryskill in the art can easily identify such variant sequences by referenceto the amino acid sequences of SEQ ID NO:2. Variant sequences can bereadily tested for functionality as described herein.

[0111] Different species can exhibit “preferential codon usage.” Ingeneral, see, Grantham et al., Nucl. Acids Res. 8:1893 (1980), Haas etal. Curr. Biol. 6:315 (1996), Wain-Hobson et al., Gene 13:355 (1981),Grosjean and Fiers, Gene 18:199 (1982), Holm, Nuc. Acids Res. 14:3075(1986), Ikemura, J. Mol. Biol. 158:573 (1982), Sharp and Matassi, Curr.Opin. Genet. Dev. 4:851 (1994), Kane, Curr. Opin. Biotechnol. 6:494(1995), and Makrides, Microbiol. Rev. 60:512 (1996). As used herein, theterm “preferential codon usage” or “preferential codons” is a term ofart referring to protein translation codons that are most frequentlyused in cells of a certain species, thus favoring one or a fewrepresentatives of the possible codons encoding each amino acid (SeeTable 2). For example, the amino acid threonine (thr) may be encoded byACA, ACC, ACG, or ACT, but in mammalian cells ACC is the most commonlyused codon; in other species, for example, insect cells, yeast, virusesor bacteria, different threonine codons may be preferential.Preferential codons for a particular species can be introduced into thepolynucleotides of the present invention by a variety of methods knownin the art. Introduction of preferential codon sequences intorecombinant DNA can, for example, enhance production of the protein bymaking protein translation more efficient within a particular cell typeor species. Therefore, the degenerate codon sequences disclosed hereinserve as a template for optimizing expression of polynucleotides invarious cell types and species commonly used in the art and disclosedherein. Sequences containing preferential codons can be tested andoptimized for expression in various species, and tested forfunctionality as disclosed herein.

[0112] The present invention further provides variant polypeptides andnucleic acid molecules that represent counterparts from other species(orthologs). These species include, but are not limited to mammalian,avian, amphibian, reptile, fish, insect and other vertebrate andinvertebrate species. As an illustration, SEQ ID NO:11, SEQ ID NO:12,and SEQ ID NO:13 provide the nucleotide, amino acid, and degeneratenucleotide sequences, respectively, of murine Zcytor 18. Features of themurine Zcytor 18 polypeptide include an extracellular domain at aminoacid residues 1 to 300 of SEQ ID NO:12, a putative signal sequence atamino acid residues 1 to 35 of SEQ ID NO:12, a transmembrane domain atamino acid residues 301 to 322 of SEQ ID NO:12, and an intracellulardomain at amino acid residues 323 to 739 of SEQ ID NO:12. Murine Zcytor18 gene expression has been detected in brain, kidney, lung, skin,testis, and uterus tissues of the mouse, while little or no expressionwas detectable in heart, liver, pancreas, and spleen tissues.

[0113] Zcytor 18 polypeptides from other mammalian species, includingmouse, porcine, ovine, bovine, canine, feline, equine, and other primatepolypeptides, are also of interest. Orthologs of human Zcytor 18 can becloned using information and compositions provided by the presentinvention in combination with conventional cloning techniques. Forexample, a Zcytor 18 cDNA can be cloned using mRNA obtained from atissue or cell type that expresses Zcytor 18 as disclosed herein.Suitable sources of mRNA can be identified by probing northern blotswith probes designed from the sequences disclosed herein. A library isthen prepared from mRNA of a positive tissue or cell line.

[0114] A Zcytor 18-encoding cDNA can be isolated by a variety ofmethods, such as by probing with a complete or partial human cDNA orwith one or more sets of degenerate probes based on the disclosedsequences. A cDNA can also be cloned using the polymerase chain reactionwith primers designed from the representative human Zcytor 18 sequencesdisclosed herein. In addition, a cDNA library can be used to transformor transfect host cells, and expression of the cDNA of interest can bedetected with an antibody to Zcytor 18 polypeptide.

[0115] Those skilled in the art will recognize that the sequencedisclosed in SEQ ID NO:1 represents a single allele of human Zcytor 18,and that allelic variation and alternative splicing are expected tooccur. Allelic variants of this sequence can be cloned by probing cDNAor genomic libraries from different individuals according to standardprocedures. Allelic variants of the nucleotide sequences disclosedherein, including those containing silent mutations and those in whichmutations result in amino acid sequence changes, are within the scope ofthe present invention, as are proteins which are allelic variants of theamino acid sequences disclosed herein. cDNA molecules generated fromalternatively spliced mRNAs, which retain the properties of the Zcytor18 polypeptide are included within the scope of the present invention,as are polypeptides encoded by such cDNAs and mRNAs. Allelic variantsand splice variants of these sequences can be cloned by probing cDNA orgenomic libraries from different individuals or tissues according tostandard procedures known in the art.

[0116] Within certain embodiments of the invention, the isolated nucleicacid molecules can hybridize under stringent conditions to nucleic acidmolecules comprising nucleotide sequences disclosed herein. For example,such nucleic acid molecules can hybridize under stringent conditions tonucleic acid molecules comprising the nucleotide sequence of SEQ IDNO:1, to nucleic acid molecules consisting of the nucleotide sequence ofnucleotides 192 to 652 of SEQ ID NO:1, nucleotide sequence ofnucleotides 192 to 2344 of SEQ ID NO:1, or to nucleic acid moleculescomprising a nucleotide sequence complementary to SEQ ID NO:1, thenucleotide sequence of nucleotides 192 to 652 of SEQ ID NO:1, ornucleotides 192 to 2344 of SEQ ID NO:1. In general, stringent conditionsare selected to be about 5° C. lower than the thermal melting point (Tm)for the specific sequence at a defined ionic strength and pH. The Tm isthe temperature (under defined ionic strength and pH) at which 50% ofthe target sequence hybridizes to a perfectly matched probe.

[0117] A pair of nucleic acid molecules, such as DNA-DNA, RNA-RNA andDNA-RNA, can hybridize if the nucleotide sequences have some degree ofcomplementarity. Hybrids can tolerate mismatched base pairs in thedouble helix, but the stability of the hybrid is influenced by thedegree of mismatch. The Tm of the mismatched hybrid decreases by 1° C.for every 1-1.5% base pair mismatch. Varying the stringency of thehybridization conditions allows control over the degree of mismatch thatwill be present in the hybrid. The degree of stringency increases as thehybridization temperature increases and the ionic strength of thehybridization buffer decreases. Stringent hybridization conditionsencompass temperatures of about 5-25° C. below the Tm of the hybrid anda hybridization buffer having up to 1 M Na⁺. Higher degrees ofstringency at lower temperatures can be achieved with the addition offormamide which reduces the T_(m) of the hybrid about 1° C. for each 1%formamide in the buffer solution. Generally, such stringent conditionsinclude temperatures of 2070° C. and a hybridization buffer containingup to 6×SSC and 0-50% formamide. A higher degree of stringency can beachieved at temperatures of from 40-70° C. with a hybridization bufferhaving up to 4×SSC and from 0-50% formamide. Highly stringent conditionstypically encompass temperatures of 42-70° C. with a hybridizationbuffer having up to 1×SSC and 0-50% formamide. Different degrees ofstringency can be used during hybridization and washing to achievemaximum specific binding to the target sequence. Typically, the washesfollowing hybridization are performed at increasing degrees ofstringency to remove non-hybridized polynucleotide probes fromhybridized complexes.

[0118] The above conditions are meant to serve as a guide and it is wellwithin the abilities of one skilled in the art to adapt these conditionsfor use with a particular polypeptide hybrid. The Tm for a specifictarget sequence is the temperature (under defined conditions) at which50% of the target sequence will hybridize to a perfectly matched probesequence. Conditions that influence the Tm include, the size and basepair content of the polynucleotide probe, the ionic strength of thehybridization solution, and the presence of destabilizing agents in thehybridization solution. Numerous equations for calculating T_(m) areknown in the art, and are specific for DNA, RNA and DNA-RNA hybrids andpolynucleotide probe sequences of varying length (see, for example,Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition(Cold Spring Harbor Press 1989); Ausubel et al., (eds.), CurrentProtocols in Molecular Biology (John Wiley and Sons, Inc. 1987); Bergerand Kimmel (eds.), Guide to Molecular Cloning Techniques, (AcademicPress, Inc. 1987); and Wetmur, Crit. Rev. Biochem. Mol. Biol. 26:227(1990)). Sequence analysis software such as OLIGO 6.0 (LSR; Long Lake,Minn.) and Primer Premier 4.0 (Premier Biosoft International; Palo Alto,Calif.), as well as sites on the Internet, are available tools foranalyzing a given sequence and calculating Tm based on user definedcriteria. Such programs can also analyze a given sequence under definedconditions and identify suitable probe sequences. Typically,hybridization of longer polynucleotide sequences, >50 base pairs, isperformed at temperatures of about 20-25° C. below the calculated T_(m).For smaller probes, <50 base pairs, hybridization is typically carriedout at the T_(m) or 5-10C below. This allows for the maximum rate ofhybridization for DNA-DNA and DNA-RNA hybrids.

[0119] The length of the polynucleotide sequence influences the rate andstability of hybrid formation. Smaller probe sequences, <50 base pairs,reach equilibrium with complementary sequences rapidly, but may formless stable hybrids. Incubation times of anywhere from minutes to hourscan be used to achieve hybrid formation. Longer probe sequences come toequilibrium more slowly, but form more stable complexes even at lowertemperatures. Incubations are allowed to proceed overnight or longer.Generally, incubations are carried out for a period equal to three timesthe calculated Cot time. Cot time, the time it takes for thepolynucleotide sequences to reassociate, can be calculated for aparticular sequence by methods known in the art.

[0120] The base pair composition of polynucleotide sequence will effectthe thermal stability of the hybrid complex, thereby influencing thechoice of hybridization temperature and the ionic strength of thehybridization buffer. A-T pairs are less stable than G-C pairs inaqueous solutions containing sodium chloride. Therefore, the higher theG-C content, the more stable the hybrid. Even distribution of G and Cresidues within the sequence also contribute positively to hybridstability. In addition, the base pair composition can be manipulated toalter the Tm of a given sequence. For example, 5-methyldeoxycytidine canbe substituted for deoxycytidine and 5-bromodeoxuridine can besubstituted for thymidine to increase the T_(m), whereas7-deazz-2′-deoxyguanosine can be substituted for guanosine to reducedependence on T_(m).

[0121] The ionic concentration of the hybridization buffer also affectsthe stability of the hybrid. Hybridization buffers generally containblocking agents such as Denhardt's solution (Sigma Chemical Co., St.Louis, Mo.), denatured salmon sperm DNA, tRNA, milk powders (BLOTTO),heparin or SDS, and a Na⁺ source, such as SSC (1×SSC: 0.15 M sodiumchloride, 15 mM sodium citrate) or SSPE (1×SSPE: 1.8 M NaCl, 10 mMNaH₂PO₄, 1 mM EDTA, pH 7.7). Typically, hybridization buffers containfrom between 10 mM 1 M Na⁺. The addition of destabilizing or denaturingagents such as formamide, tetralkylammonium salts, guanidinium cationsor thiocyanate cations to the hybridization solution will alter theT_(m) of a hybrid. Typically, formamide is used at a concentration of upto 50% to allow incubations to be carried out at more convenient andlower temperatures. Formamide also acts to reduce non-specificbackground when using RNA probes.

[0122] As an illustration, a nucleic acid molecule encoding a variantZcytor 18 polypeptide can be hybridized with a nucleic acid moleculehaving the nucleotide sequence of SEQ ID NO:1 (or its complement) at 42°C. overnight in a solution comprising 50% formamide, 5×SSC, 50 mM sodiumphosphate (pH 7.6), 5× Denhardt's solution (100× Denhardt's solution: 2%(w/v) Ficoll 400, 2% (w/v) polyvinylpyrrolidone, and 2% (w/v) bovineserum albumin), 10% dextran sulfate, and 20 μg/ml denatured, shearedsalmon sperm DNA. One of skill in the art can devise variations of thesehybridization conditions. For example, the hybridization mixture can beincubated at a higher temperature, such as about 65° C., in a solutionthat does not contain formamide. Moreover, premixed hybridizationsolutions are available (e.g., EXPRESSHYB Hybridization Solution fromCLONTECH Laboratories, Inc.), and hybridization can be performedaccording to the manufacturer's instructions.

[0123] Following hybridization, the nucleic acid molecules can be washedto remove non-hybridized nucleic acid molecules under stringentconditions, or under highly stringent conditions. Typical stringentwashing conditions include washing in a solution of 0.5×-2×SSC with 0.1%sodium dodecyl sulfate (SDS) at 55-65° C. As an illustration, nucleicacid molecules encoding a variant Zcytor 18 polypeptide remainhybridized with a nucleic acid molecule comprising the nucleotidesequence of nucleotides 192 to 652 of SEQ ID NO:1 (or its complement)under stringent washing conditions, in which the wash stringency isequivalent to 0.5×-2×SSC with 0.1% SDS at 55-65° C., including 0.5×SSCwith 0.1% SDS at 55° C., or 2×SSC with 0.1% SDS at 65° C. One of skillin the art can readily devise equivalent conditions, for example, bysubstituting SSPE for SSC in the wash solution.

[0124] Typical highly stringent washing conditions include washing in asolution of 0.1×-0.2×SSC with 0.1% sodium dodecyl sulfate (SDS) at50-65° C. For example, nucleic acid molecules encoding a variant Zcytor18 polypeptide remain hybridized with a nucleic acid molecule comprisingthe nucleotide sequence of nucleotides 192 to 652 of SEQ ID NO:1 (or itscomplement) under highly stringent washing conditions, in which the washstringency is equivalent to 0.1×-0.2×SSC with 0.1% SDS at 50-65° C.,including 0.1×SSC with 0.1% SDS at 50° C., or 0.2×SSC with 0.1% SDS at65° C.

[0125] The present invention also provides isolated Zcytor 18polypeptides that have a substantially similar sequence identity to thepolypeptides of SEQ ID NO:2, SEQ ID NO:8, or their orthologs. The term“substantially similar sequence identity” is used herein to denotepolypeptides having at least 70%, at least 80%, at least 90%, at least95% or greater than 95% sequence identity to the sequences shown in SEQID NO:2, SEQ ID NO:8, or their orthologs.

[0126] The present invention also contemplates Zcytor 18 variant nucleicacid molecules that can be identified using two criteria: adetermination of the similarity between the encoded polypeptide with theamino acid sequence of SEQ ID NO:2, and a hybridization assay, asdescribed above. Such Zcytor 18 variants include nucleic acid molecules(1) that remain hybridized with a nucleic acid molecule comprising thenucleotide sequence of nucleotides 192 to 652 of SEQ ID NO:1 (or itscomplement) under stringent washing conditions, in which the washstringency is equivalent to 0.5×2×SSC with 0.1% SDS at 55-65° C., and(2) that encode a polypeptide having at least 70%, at least 80%, atleast 90%, at least 95% or greater than 95% sequence identity to theamino acid sequence of SEQ ID NO:2. Alternatively, Zcytor 18 variantscan be characterized as nucleic acid molecules (1) that remainhybridized with a nucleic acid molecule comprising the nucleotidesequence of nucleotides 192 to 652 of SEQ ID NO:1 (or its complement)under highly stringent washing conditions, in which the wash stringencyis equivalent to 0.1×-0.2×SSC with 0.1% SDS at 50-65° C., and (2) thatencode a polypeptide having at least 70%, at least 80%, at least 90%, atleast 95% or greater than 95% sequence identity to the amino acidsequence of SEQ ID NO:2.

[0127] Percent sequence identity is determined by conventional methods.See, for example, Altschul et al., Bull. Math. Bio. 48:603 (1986), andHenikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1992).Briefly, two amino acid sequences are aligned to optimize the alignmentscores using a gap opening penalty of 10, a gap extension penalty of 1,and the “BLOSUM62” scoring matrix of Henikoff and Henikoff (ibid.) asshown in Table 3 (amino acids are indicated by the standard one-lettercodes). The percent identity is then calculated as: ([Total number ofidentical matches]/[length of the longer sequence plus the number ofgaps introduced into the longer sequence in order to align the twosequences])(100). TABLE 3 A R N D C Q E G H I L K M F P S T W Y V A 4 R−1 5 N −2 0 6 D −2 −2 1 6 C 0 −3 −3 −3 9 Q −1 1 0 0 −3 5 E −1 0 0 2 −4 25 G 0 −2 0 −1 −3 −2 −2 6 H −2 0 1 −1 −3 0 0 −2 8 I −1 −3 −3 −3 −1 −3 −3−4 −3 4 L −1 −2 −3 −4 −1 −2 −3 −4 −3 2 4 K −1 2 0 −1 −3 1 1 −2 −1 −3 −25 M −1 −1 −2 −3 −1 0 −2 −3 −2 1 2 −1 5 F −2 −3 −3 −3 −2 −3 −3 −3 −1 0 0−3 0 6 P −1 −2 −2 −1 −3 −1 −1 −2 −2 −3 −3 −1 −2 −4 7 S 1 −1 1 0 −1 0 0 0−1 −2 −2 0 −1 −2 −1 4 T 0 −1 0 −1 −1 −1 −1 −2 −2 −1 −1 −1 −1 −2 −1 1 5 W−3 −3 −4 −4 −2 −2 −3 −2 −2 −3 −2 −3 −1 1 −4 −3 −2 11 Y −2 −2 −2 −3 −2 −1−2 −3 2 −1 −1 −2 −1 3 −3 −2 −2 2 7 V 0 −3 −3 −3 −1 −2 −2 −3 −3 3 1 −2 1−1 −2 −2 0 −3 −1 4

[0128] Those skilled in the art appreciate that there are manyestablished algorithms available to align two amino acid sequences. The“FASTA” similarity search algorithm of Pearson and Lipman is a suitableprotein alignment method for examining the level of identity shared byan amino acid sequence disclosed herein and the amino acid sequence of aputative Zcytor 18 variant. The FASTA algorithm is described by Pearsonand Lipman, Proc. Nat'l Acad. Sci. USA 85:2444 (1988), and by Pearson,Meth. Enzymol. 183:63 (1990). Briefly, FASTA first characterizessequence similarity by identifying regions shared by the query sequence(e.g., SEQ ID NO:2) and a test sequence that have either the highestdensity of identities (if the ktup variable is 1) or pairs of identities(if ktup=2), without considering conservative amino acid substitutions,insertions, or deletions. The ten regions with the highest density ofidentities are then rescored by comparing the similarity of all pairedamino acids using an amino acid substitution matrix, and the ends of theregions are “trimmed” to include only those residues that contribute tothe highest score. If there are several regions with scores greater thanthe “cutoff” value (calculated by a predetermined formula based upon thelength of the sequence and the ktup value), then the trimmed initialregions are examined to determine whether the regions can be joined toform an approximate alignment with gaps. Finally, the highest scoringregions of the two amino acid sequences are aligned using a modificationof the Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol.Biol. 48:444 (1970); Sellers, SIAM J. Appl. Math. 26:787 (1974)), whichallows for amino acid insertions and deletions. Illustrative parametersfor FASTA analysis are: ktup=1, gap opening penalty=10, gap extensionpenalty=1, and substitution matrix=BLOSUM62. These parameters can beintroduced into a FASTA program by modifying the scoring matrix file(“SMATRIX”), as explained in Appendix 2 of Pearson, Meth. Enzymol.183:63 (1990).

[0129] FASTA can also be used to determine the sequence identity ofnucleic acid molecules using a ratio as disclosed above. For nucleotidesequence comparisons, the ktup value can range between one to six,preferably from three to six, most preferably three, with otherparameters set as described above.

[0130] The present invention includes nucleic acid molecules that encodea polypeptide having a conservative amino acid change, compared with anamino acid sequence disclosed herein. For example, variants can beobtained that contain one or more amino acid substitutions of SEQ IDNO:2, or SEQ ID NO:8, in which an alkyl amino acid is substituted for analkyl amino acid in a Zcytor 18 amino acid sequence, an aromatic aminoacid is substituted for an aromatic amino acid in a Zcytor 18 amino acidsequence, a sulfur-containing amino acid is substituted for asulfur-containing amino acid in a Zcytor 18 amino acid sequence, ahydroxy-containing amino acid is substituted for a hydroxy-containingamino acid in a Zcytor 18 amino acid sequence, an acidic amino acid issubstituted for an acidic amino acid in a Zcytor 18 amino acid sequence,a basic amino acid is substituted for a basic amino acid in a Zcytor 18amino acid sequence, or a dibasic monocarboxylic amino acid issubstituted for a dibasic monocarboxylic amino acid in a Zcytor 18 aminoacid sequence. Among the common amino acids, for example, a“conservative amino acid substitution” is illustrated by a substitutionamong amino acids within each of the following groups: (1) glycine,alanine, valine, leucine, and isoleucine, (2) phenylalanine, tyrosine,and tryptophan, (3) serine and threonine, (4) aspartate and glutamate,(5) glutamine and asparagine, and (6) lysine, arginine and histidine.

[0131] The BLOSUM62 table is an amino acid substitution matrix derivedfrom about 2,000 local multiple alignments of protein sequence segments,representing highly conserved regions of more than 500 groups of relatedproteins (Henikoff and Henikoff, Proc. Nat'l Acad. Sci. USA 89:10915(1992)). Accordingly, the BLOSUM62 substitution frequencies can be usedto define conservative amino acid substitutions that may be introducedinto the amino acid sequences of the present invention. Although it ispossible to design amino acid substitutions based solely upon chemicalproperties (as discussed above), the language “conservative amino acidsubstitution” preferably refers to a substitution represented by aBLOSUM62 value of greater than −1. For example, an amino acidsubstitution is conservative if the substitution is characterized by aBLOSUM62 value of 0, 1, 2, or 3. According to this system, preferredconservative amino acid substitutions are characterized by a BLOSUM62value of at least 1 (e.g., 1, 2 or 3), while more preferred conservativeamino acid substitutions are characterized by a BLOSUM62 value of atleast 2 (e.g., 2 or 3).

[0132] Particular variants of Zcytor 18 are characterized by having atleast 70%, at least 80%, at least 90%, at least 95% or greater than 95%sequence identity to the corresponding amino acid sequence (e.g., SEQ IDNO:2 or SEQ ID NO:8), wherein the variation in amino acid sequence isdue to one or more conservative amino acid substitutions.

[0133] Conservative amino acid changes in a Zcytor 18 gene can beintroduced, for example, by substituting nucleotides for the nucleotidesrecited in SEQ ID NO:1. Such “conservative amino acid” variants can beobtained by oligonucleotide-directed mutagenesis, linker-scanningmutagenesis, mutagenesis using the polymerase chain reaction, and thelike (see Ausubel (1995) at pages 8-10 to 8-22; and McPherson (ed.),Directed Mutagenesis: A Practical Approach (IRL Press 1991)). A variantZcytor 18 polypeptide can be identified by the ability to specificallybind anti-Zcytor 18 antibodies.

[0134] The proteins of the present invention can also comprisenon-naturally occurring amino acid residues. Non-naturally occurringamino acids include, without limitation, trans-3-methylproline,2,4-methanoproline, cis-4-hydroxyproline, trans-4hydroxyproline,N-methylglycine, allo-threonine, methylthreonine, hydroxyethylcysteine,hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid,thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline,3,3-dimethylproline, tert-leucine, norvaline, 2-azaphenylalanine,3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine.Several methods are known in the art for incorporating non-naturallyoccurring amino acid residues into proteins. For example, an in vitrosystem can be employed wherein nonsense mutations are suppressed usingchemically aminoacylated suppressor tRNAs. Methods for synthesizingamino acids and aminoacylating tRNA are known in the art. Transcriptionand translation of plasmids containing nonsense mutations is typicallycarried out in a cell-free system comprising an E. coli S30 extract andcommercially available enzymes and other reagents. Proteins are purifiedby chromatography. See, for example, Robertson et al., J. Am. Chem. Soc.113:2722 (1991), Ellman et al., Methods Enzymol. 202:301 (1991), Chunget al., Science 259:806 (1993), and Chung et al., Proc. Nat'l Acad. Sci.USA 90:10145 (1993).

[0135] In a second method, translation is carried out in Xenopus oocytesby microinjection of mutated mRNA and chemically aminoacylatedsuppressor tRNAs (Turcatti et al., J. Biol. Chem. 271:19991 (1996)).Within a third method, E. coli cells are cultured in the absence of anatural amino acid that is to be replaced (e.g., phenylalanine) and inthe presence of the desired non-naturally occurring amino acid(s) (e.g.,2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or0.4fluorophenylalanine). The non-naturally occurring amino acid isincorporated into the protein in place of its natural counterpart. See,Koide et al., Biochem. 33:7470 (1994). Naturally occurring amino acidresidues can be converted to non-naturally occurring species by in vitrochemical modification. Chemical modification can be combined withsite-directed mutagenesis to further expand the range of substitutions(Wynn and Richards, Protein Sci. 2:395 (1993)).

[0136] A limited number of non-conservative amino acids, amino acidsthat are not encoded by the genetic code, non-naturally occurring aminoacids, and unnatural amino acids may be substituted for Zcytor 18 aminoacid residues.

[0137] Essential amino acids in the polypeptides of the presentinvention can be identified according to procedures known in the art,such as site-directed mutagenesis or alanine-scanning mutagenesis(Cunningham and Wells, Science 244:1081 (1989), Bass et al., Proc. Nat'lAcad. Sci. USA 88:4498 (1991), Coombs and Corey, “Site-DirectedMutagenesis and Protein Engineering,” in Proteins: Analysis and Design,Angeletti (ed.), pages 259-311 (Academic Press, Inc. 1998)). In thelatter technique, single alanine mutations are introduced at everyresidue in the molecule, and the resultant mutant molecules are testedfor biological activity to identify amino acid residues that arecritical to the activity of the molecule. See also, Hilton et al., J.Biol. Chem. 271:4699 (1996).

[0138] Although sequence analysis can be used to further define theZcytor 18 ligand binding region, amino acids that play a role in Zcytor18 binding activity can also be determined by physical analysis ofstructure, as determined by such techniques as nuclear magneticresonance, crystallography, electron diffraction or photoaffinitylabeling, in conjunction with mutation of putative contact site aminoacids. See, for example, de Vos et al., Science 255:306 (1992), Smith etal., J. Mol. Biol. 224:899 (1992), and Wlodaver et al., FEBS Lett.309:59 (1992).

[0139] Multiple amino acid substitutions can be made and tested usingknown methods of mutagenesis and screening, such as those disclosed byReidhaar-Olson and Sauer (Science 241:53 (1988)) or Bowie and Sauer(Proc. Nat'l Acad. Sci. USA 86:2152 (1989)). Briefly, these authorsdisclose methods for simultaneously randomizing two or more positions ina polypeptide, selecting for functional polypeptide, and then sequencingthe mutagenized polypeptides to determine the spectrum of allowablesubstitutions at each position. Other methods that can be used includephage display (e.g., Lowman et al., Biochem. 30:10832 (1991), Ladner etal., U.S. Pat. No. 5,223,409, Huse, international publication No. WO92/06204, and region-directed mutagenesis (Derbyshire et al., Gene46:145 (1986), and Ner et al., DNA 7:127, (1988)). Moreover, Zcytor 18labeled with biotin or FITC can be used for expression cloning of Zcytor18 ligands.

[0140] Variants of the disclosed Zcytor 18 nucleotide and polypeptidesequences can also be generated through DNA shuffling as disclosed byStemmer, Nature 370:389 (1994), Stemmer, Proc. Nat'l Acad. Sci. USA91:10747 (1994), and international publication No. WO 97/20078. Briefly,variant DNA molecules are generated by in vitro homologous recombinationby random fragmentation of a parent DNA followed by reassembly usingPCR, resulting in randomly introduced point mutations. This techniquecan be modified by using a family of parent DNA molecules, such asallelic variants or DNA molecules from different species, to introduceadditional variability into the process. Selection or screening for thedesired activity, followed by additional iterations of mutagenesis andassay provides for rapid “evolution” of sequences by selecting fordesirable mutations while simultaneously selecting against detrimentalchanges.

[0141] Mutagenesis methods as disclosed herein can be combined withhigh-throughput, automated screening methods to detect activity ofcloned, mutagenized polypeptides in host cells. Mutagenized DNAmolecules that encode biologically active polypeptides, or polypeptidesthat bind with anti-Zcytor 18 antibodies, can be recovered from the hostcells and rapidly sequenced using modern equipment. These methods allowthe rapid determination of the importance of individual amino acidresidues in a polypeptide of interest, and can be applied topolypeptides of unknown structure.

[0142] The present invention also includes “functional fragments” ofZcytor 18 polypeptides and nucleic acid molecules encoding suchfunctional fragments. Routine deletion analyses of nucleic acidmolecules can be performed to obtain functional fragments of a nucleicacid molecule that encodes a Zcytor 18 polypeptide. As an illustration,DNA molecules comprising the nucleotide sequence of nucleotides 192 to2347 of SEQ ID NO:1 can be digested with Bal31 nuclease to obtain aseries of nested deletions. The fragments are then inserted intoexpression vectors in proper reading frame, and the expressedpolypeptides are isolated and tested for the ability to bind anti-Zcytor18 antibodies. One alternative to exonuclease digestion is to useoligonucleotide-directed mutagenesis to introduce deletions or stopcodons to specify production of a desired fragment. Alternatively,particular fragments of a Zcytor 18 gene can be synthesized using thepolymerase chain reaction. An example of a functional fragment is theextracellular domain of Zcytor 18 (i.e., amino acid residues 36 to 313of SEQ ID NO:2 or SEQ ID NO:5, or amino acid residues 36 to 299 of SEQID NO:8).

[0143] This general approach is exemplified by studies on the truncationat either or both termini of interferons have been summarized byHorisberger and Di Marco, Pharmac. Ther. 66:507 (1995). Moreover,standard techniques for functional analysis of proteins are describedby, for example, Treuter et al., Molec. Gen. Genet. 240:113 (1993),Content et al., “Expression and preliminary deletion analysis of the 42kDa 2-SA synthetase induced by human interferon,” in BiologicalInterferon Systems, Proceedings of ISIR-TNO Meeting on InterferonSystems, Cantell (ed.), pages 65-72 (Nijhoff 1987), Herschman, “The EGFReceptor,” in Control of Animal Cell Proliferation, Vol. 1, Boynton etal., (eds.) pages 169-199 (Academic Press 1985), Coumailleau et al., J.Biol. Chem. 270:29270 (1995); Fukunaga et al., J. Biol. Chem. 270:25291(1995); Yamaguchi et al., Biochem. Pharmacol. 50:1295 (1995), and Meiselet al., Plant Molec. Biol. 30:1 (1996).

[0144] The present invention also contemplates functional fragments of aZcytor 18 gene that have amino acid changes, compared with an amino acidsequence disclosed herein. A variant Zcytor 18 gene can be identified onthe basis of structure by determining the level of identity withdisclosed nucleotide and amino acid sequences, as discussed above. Analternative approach to identifying a variant gene on the basis ofstructure is to determine whether a nucleic acid molecule encoding apotential variant Zcytor 18 gene can hybridize to a nucleic acidmolecule comprising a nucleotide sequence, such as SEQ ID NO:1.

[0145] The present invention also provides polypeptide fragments orpeptides comprising an epitope-bearing portion of a Zcytor 18polypeptide described herein. Such fragments or peptides may comprise an“immunogenic epitope,” which is a part of a protein that elicits anantibody response when the entire protein is used as an immunogen.Immunogenic epitope-bearing peptides can be identified using standardmethods (see, for example, Geysen et al., Proc. Nat'l Acad. Sci. USA81:3998 (1983)).

[0146] In contrast, polypeptide fragments or peptides may comprise an“antigenic epitope,” which is a region of a protein molecule to which anantibody can specifically bind. Certain epitopes consist of a linear orcontiguous stretch of amino acids, and the antigenicity of such anepitope is not disrupted by denaturing agents. It is known in the artthat relatively short synthetic peptides that can mimic epitopes of aprotein can be used to stimulate the production of antibodies againstthe protein (see, for example, Sutcliffe et al., Science 219:660(1983)).)). Antibodies that recognize short linear epitopes areparticularly useful in analytic and diagnostic applications that usedenatured protein, such as Western analysis, or in the analysis of fixedcells or tissue samples. Antibodies to linear epitopes are also usefulfor detecting fragments of Zcytor 18, such as might occur in body fluidsor culture media. Accordingly, antigenic epitope-bearing peptides andpolypeptides of the present invention are useful to raise antibodiesthat bind with the polypeptides described herein.

[0147] Antigenic epitope-bearing peptides and polypeptides can containat least four to ten amino acids, at least ten to fifteen amino acids,or about 15 to about 30 amino acids of an amino acid sequence disclosedherein. Such epitope-bearing peptides and polypeptides can be producedby fragmenting a Zcytor 18 polypeptide, or by chemical peptidesynthesis, as described herein. Moreover, epitopes can be selected byphage display of random peptide libraries (see, for example, Lane andStephen, Curr. Opin. Immunol. 5:268 (1993), and Cortese et al., Curr.Opin. Biotechnol. 7:616 (1996)). Standard methods for identifyingepitopes and producing antibodies from small peptides that comprise anepitope are described, for example, by Mole, “Epitope Mapping,” inMethods in Molecular Biology, Vol. 10, Manson (ed.), pages 105-116 (TheHumana Press, Inc. 1992), Price, “Production and Characterization ofSynthetic Peptide-Derived Antibodies,” in Monoclonal Antibodies:Production, Engineering, and Clinical Application, Ritter and Ladyman(eds.), pages 60-84 (Cambridge University Press 1995), and Coligan etal. (eds.), Current Protocols in Immunology, pages 9.3.1-9.3.5 and pages9.4.1-9.4.11 (John Wiley & Sons 1997).

[0148] For any Zcytor 18 polypeptide, including variants and fusionproteins, one of ordinary skill in the art can readily generate a fullydegenerate polynucleotide sequence encoding that variant using theinformation set forth in Tables 1 and 2 above. Moreover, those of skillin the art can use standard software to devise Zcytor 18 variants basedupon the nucleotide and amino acid sequences described herein.Accordingly, the present invention includes a computer-readable mediumencoded with a data structure that provides at least one of thefollowing sequences: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4,SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9.Suitable forms of computer-readable media include magnetic media andoptically-readable media. Examples of magnetic media include a hard orfixed drive, a random access memory (RAM) chip, a floppy disk, digitallinear tape (DLT), a disk cache, and a ZIP disk. Optically readablemedia are exemplified by compact discs (e.g., CD-read only memory (ROM),CD-rewritable (RW), and CDrecordable), and digital versatile/video discs(DVD) (e.g., DVD-ROM, DVD-RAM, and DVD+RW).

[0149] 5. Production of Zcytor 18 Polypeptides

[0150] The polypeptides of the present invention, including full-lengthpolypeptides, functional fragments, and fusion proteins, can be producedin recombinant host cells following conventional techniques. To expressa Zcytor 18 gene, a nucleic acid molecule encoding the polypeptide mustbe operably linked to regulatory sequences that control transcriptionalexpression in an expression vector and then, introduced into a hostcell. In addition to transcriptional regulatory sequences, such aspromoters and enhancers, expression vectors can include translationalregulatory sequences and a marker gene which is suitable for selectionof cells that carry the expression vector.

[0151] Expression vectors that are suitable for production of a foreignprotein in eukaryotic cells typically contain (1) prokaryotic DNAelements coding for a bacterial replication origin and an antibioticresistance marker to provide for the growth and selection of theexpression vector in a bacterial host; (2) eukaryotic DNA elements thatcontrol initiation of transcription, such as a promoter; and (3) DNAelements that control the processing of transcripts, such as atranscription termination/polyadenylation sequence. As discussed above,expression vectors can also include nucleotide sequences encoding asecretory sequence that directs the heterologous polypeptide into thesecretory pathway of a host cell. For example, a Zcytor 18 expressionvector may comprise a Zcytor 18 gene and a secretory sequence derivedfrom any secreted gene.

[0152] Zcytor 18 proteins of the present invention may be expressed inmammalian cells. Examples of suitable mammalian host cells includeAfrican green monkey kidney cells (Vero; ATCC CRL 1587), human embryonickidney cells (293HEK; ATCC CRL 1573), baby hamster kidney cells (BHK-21,BHK-570; ATCC CRL 8544, ATCC CRL 10314), canine kidney cells (MDCK; ATCCCCL 34), Chinese hamster ovary cells (CHO-KI; ATCC CCL61; CHO DG44(Chasin et al., Som. Cell. Molec. Genet. 12:555, 1986)), rat pituitarycells (GH1; ATCC CCL82), HeLa S3 cells (ATCC CCL2.2), rat hepatoma cells(H-4-II-E; ATCC CRL 1548) SV40-transformed monkey kidney cells (COS-1;ATCC CRL 1650) and murine embryonic cells (NIH3T3; ATCC CRL 1658).

[0153] For a mammalian host, the transcriptional and translationalregulatory signals may be derived from viral sources, such asadenovirus, bovine papilloma virus, simian virus, or the like, in whichthe regulatory signals are associated with a particular gene which has ahigh level of expression. Suitable transcriptional and translationalregulatory sequences also can be obtained from mammalian genes, such asactin, collagen, myosin, and metallothionein genes.

[0154] Transcriptional regulatory sequences include a promoter regionsufficient to direct the initiation of RNA synthesis. Suitableeukaryotic promoters include the promoter of the mouse metallothionein Igene (Hamer et al., J. Molec. Appl. Genet. 1:273 (1982)), the TKpromoter of Herpes virus (McKnight, Cell 31:355 (1982)), the SV40 earlypromoter (Benoist et al., Nature 290:304 (1981)), the Rous sarcoma viruspromoter (Gorman et al., Proc. Nat'l Acad. Sci. USA 79:6777 (1982)), thecytomegalovirus promoter (Foecking et al., Gene 45:101 (1980)), and themouse mammary tumor virus promoter (see, generally, Etcheverry,“Expression of Engineered Proteins in Mammalian Cell Culture,” inProtein Engineering: Principles and Practice, Cleland et al. (eds.),pages 163-181 (John Wiley & Sons, Inc. 1996)).

[0155] Alternatively, a prokaryotic promoter, such as the bacteriophageT3 RNA polymerase promoter, can be used to control Zcytor 18 geneexpression in mammalian cells if the prokaryotic promoter is regulatedby a eukaryotic promoter (Zhou et al., Mol. Cell. Biol. 10:4529 (1990),and Kaufman et al., Nucl. Acids Res. 19:4485 (1991)).

[0156] An expression vector can be introduced into host cells using avariety of standard techniques including calcium phosphate transfection,liposome-mediated transfection, microprojectile-mediated delivery,electroporation, and the like. The transfected cells can be selected andpropagated to provide recombinant host cells that comprise theexpression vector stably integrated in the host cell genome. Techniquesfor introducing vectors into eukaryotic cells and techniques forselecting such stable transformants using a dominant selectable markerare described, for example, by Ausubel (1995) and by Murray (ed.), GeneTransfer and Expression Protocols (Humana Press 1991).

[0157] For example, one suitable selectable marker is a gene thatprovides resistance to the antibiotic neomycin. In this case, selectionis carried out in the presence of a neomycin-type drug, such as G-418 orthe like. Selection systems can also be used to increase the expressionlevel of the gene of interest, a process referred to as “amplification.”Amplification is carried out by culturing transfectants in the presenceof a low level of the selective agent and then increasing the amount ofselective agent to select for cells that produce high levels of theproducts of the introduced genes. A suitable amplifiable selectablemarker is dihydrofolate reductase, which confers resistance tomethotrexate. Other drug resistance genes (e.g., hygromycin resistance,multi-drug resistance, puromycin acetyltransferase) can also be used.Alternatively, markers that introduce an altered phenotype, such asgreen fluorescent protein, or cell surface proteins such as CD4, CD8,Class I MHC, placental alkaline phosphatase may be used to sorttransfected cells from untransfected cells by such means as FACS sortingor magnetic bead separation technology.

[0158] Zcytor 18 polypeptides can also be produced by cultured mammaliancells using a viral delivery system. Exemplary viruses for this purposeinclude adenovirus, herpesvirus, vaccinia virus and adeno-associatedvirus (AAV). Adenovirus, a double-stranded DNA virus, is currently thebest studied gene transfer vector for delivery of heterologous nucleicacid (for a review, see Becker et al., Meth. Cell Biol. 43:161 (1994),and Douglas and Curiel, Science & Medicine 4:44 (1997)). Advantages ofthe adenovirus system include the accommodation of relatively large DNAinserts, the ability to grow to high-titer, the ability to infect abroad range of mammalian cell types, and flexibility that allows usewith a large number of available vectors containing different promoters.

[0159] By deleting portions of the adenovirus genome, larger inserts (upto 7 kb) of heterologous DNA can be accommodated. These inserts can beincorporated into the viral DNA by direct ligation or by homologousrecombination with a co-transfected plasmid. An option is to delete theessential E1 gene from the viral vector, which results in the inabilityto replicate unless the E1 gene is provided by the host cell. Adenovirusvector-infected human 293 cells (ATCC Nos. CRL-1573, 45504, 45505), forexample, can be grown as adherent cells or in suspension culture atrelatively high cell density to produce significant amounts of protein(see Garnier et al., Cytotechnol. 15:145 (1994)).

[0160] Zcytor 18 can also be expressed in other higher eukaryotic cells,such as avian, fungal, insect, yeast, or plant cells. The baculovirussystem provides an efficient means to introduce cloned Zcytor 18 genesinto insect cells. Suitable expression vectors are based upon theAutographa californica multiple nuclear polyhedrosis virus (AcMNPV), andcontain well-known promoters such as Drosophila heat shock protein (hsp)70 promoter, Autographa californica nuclear polyhedrosis virusimmediate-early gene promoter (ie-1) and the delayed early 39K promoter,baculovirus p10 promoter, and the Drosophila metallothionein promoter. Asecond method of making recombinant baculovirus utilizes atransposon-based system described by Luckow (Luckow, et al., J. Virol.67:4566 (1993)). This system, which utilizes transfer vectors, is soldin the BAC-to-BAC kit (Life Technologies, Rockville, Md.). This systemutilizes a transfer vector, PFASTBAC (Life Technologies) containing aTn7 transposon to move the DNA encoding the Zcytor 18 polypeptide into abaculovirus genome maintained in E. coli as a large plasmid called a“bacmid.” See, Hill-Perkins and Possee, J. Gen. Virol. 71:971 (1990),Bonning, et al., J. Gen. Virol. 75:1551 (1994), and Chazenbalk, andRapoport, J. Biol. Chem. 270:1543 (1995). In addition, transfer vectorscan include an in-frame fusion with DNA encoding an epitope tag at theC- or N-terminus of the expressed Zcytor 18 polypeptide, for example, aGlu-Glu epitope tag (Grussenmeyer et al., Proc. Nat'l Acad. Sci. 82:7952(1985)). Using a technique known in the art, a transfer vectorcontaining a Zcytor 18 gene is transformed into E. coli, and screenedfor bacmids, which contain an interrupted lacZ gene indicative ofrecombinant baculovirus. The bacmid DNA containing the recombinantbaculovirus genome is then isolated using common techniques.

[0161] The illustrative PFASTBAC vector can be modified to aconsiderable degree. For example, the polyhedrin promoter can be removedand substituted with the baculovirus basic protein promoter (also knownas Pcor, p6.9 or MP promoter) which is expressed earlier in thebaculovirus infection, and has been shown to be advantageous forexpressing secreted proteins (see, for example, Hill-Perkins and Possee,J. Gen. Virol. 71:971 (1990), Bonning, et al., J. Gen. Virol. 75:1551(1994), and Chazenbalk and Rapoport, J. Biol. Chem. 270:1543 (1995). Insuch transfer vector constructs, a short or long version of the basicprotein promoter can be used. Moreover, transfer vectors can beconstructed, which replace the native Zcytor 18 secretory signalsequences with secretory signal sequences derived from insect proteins.For example, a secretory signal sequence from EcdysteroidGlucosyltransferase (EGT), honey bee Melittin (Invitrogen Corporation;Carlsbad, Calif.), or baculovirus gp67 (PharMingen: San Diego, Calif.)can be used in constructs to replace the native Zcytor 18 secretorysignal sequence.

[0162] The recombinant virus or bacmid is used to transfect host cells.Suitable insect host cells include cell lines derived from IPLB-Sf-21, aSpodoptera frugiperda pupal ovarian cell line, such as SJ9 (ATCC CRL1711), SJ21AE, and SJ21 (Invitrogen Corporation; San Diego, Calif.), aswell as Drosophila Schneider-2 cells, and the HIGH FIVEO cell line(Invitrogen) derived from Trichoplusia ni (U.S. Pat. No. 5,300,435).Commercially available serum-free media can be used to grow and tomaintain the cells. Suitable media are Sf900 IIM (Life Technologies) orESF 921™ (Expression Systems) for the Sf9 cells; and Ex-cellO405™ (JRHBiosciences, Lenexa, Kans.) or Express FiveO™ (Life Technologies) forthe T. ni cells. When recombinant virus is used, the cells are typicallygrown up from an inoculation density of approximately 2-5×10⁵ cells to adensity of 1-2×10⁶ cells at which time a recombinant viral stock isadded at a multiplicity of infection (MOI) of 0.1 to 10, more typicallynear 3.

[0163] Established techniques for producing recombinant proteins inbaculovirus systems are provided by Bailey et al., “Manipulation ofBaculovirus Vectors,” in Methods in Molecular Biology, Volume 7: GeneTransfer and Expression Protocols, Murray (ed.), pages 147-168 (TheHumana Press, Inc. 1991), by Patel et al., “The baculovirus expressionsystem,” in DNA Cloning 2: Expression Systems, 2nd Edition, Glover etal. (eds.), pages 205-244 (Oxford University Press 1995), by Ausubel(1995) at pages 16-37 to 16-57, by Richardson (ed.), BaculovirusExpression Protocols (The Humana Press, Inc. 1995), and by Lucknow,“Insect Cell Expression Technology,” in Protein Engineering: Principlesand Practice, Cleland et al. (eds.), pages 183-218 (John Wiley & Sons,Inc. 1996).

[0164] Fungal cells, including yeast cells, can also be used to expressthe genes described herein. Yeast species of particular interest in thisregard include Saccharomyces cerevisiae, Pichia pastoris, and Pichiamethanolica. Suitable promoters for expression in yeast includepromoters from GAL1 (galactose), PGK (phosphoglycerate kinase), ADH(alcohol dehydrogenase), AOX1 (alcohol oxidase), HIS4 (histidinoldehydrogenase), and the like. Many yeast cloning vectors have beendesigned and are readily available. These vectors include YIp-basedvectors, such as YIp5, YRp vectors, such as YRp17, YEp vectors such asYEpl3 and YCp vectors, such as YCp19. Methods for transforming S.cerevisiae cells with exogenous DNA and producing recombinantpolypeptides therefrom are disclosed by, for example, Kawasaki, U.S.Pat. No. 4,599,311, Kawasaki et al., U.S. Pat. No. 4,931,373, Brake,U.S. Pat. No. 4,870,008, Welch et al., U.S. Pat. No. 5,037,743, andMurray et al., U.S. Pat. No. 4,845,075. Transformed cells are selectedby phenotype determined by the selectable marker, commonly drugresistance or the ability to grow in the absence of a particularnutrient (e.g., leucine). A suitable vector system for use inSaccharomyces cerevisiae is the POT1 vector system disclosed by Kawasakiet al. (U.S. Pat. No. 4,931,373), which allows transformed cells to beselected by growth in glucose-containing media. Additional suitablepromoters and terminators for use in yeast include those from glycolyticenzyme genes (see, e.g., Kawasaki, U.S. Pat. No. 4,599,311, Kingsman etal., U.S. Pat. No. 4,615,974, and Bitter, U.S. Pat. No. 4,977,092) andalcohol dehydrogenase genes. See also U.S. Pat. Nos. 4,990,446,5,063,154, 5,139,936, and 4,661,454.

[0165] Transformation systems for other yeasts, including Hansenulapolymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis,Kluyveromyces fragilis, Ustilago maydis, Pichia pastoris, Pichiamethanolica, Pichia guillermondii and Candida maltosa are known in theart. See, for example, Gleeson et al., J. Gen. Microbiol. 132:3459(1986), and Cregg, U.S. Pat. No. 4,882,279. Aspergillus cells may beutilized according to the methods of McKnight et al., U.S. Pat. No.4,935,349. Methods for transforming Acremonium chrysogenum are disclosedby Sumino et al., U.S. Pat. No. 5,162,228. Methods for transformingNeurospora are disclosed by Lambowitz, U.S. Pat. No. 4,486,533.

[0166] For example, the use of Pichia methanolica as host for theproduction of recombinant proteins is disclosed by Raymond, U.S. Pat.No. 5,716,808, Raymond, U.S. Pat. No. 5,736,383, Raymond et al., Yeast14:11-23 (1998), and in international publication Nos. WO 97/17450, WO97/17451, WO 98/02536, and WO 98/02565. DNA molecules for use intransforming P. methanolica will commonly be prepared asdouble-stranded, circular plasmids, which can be linearized prior totransformation. For polypeptide production in P. methanolica, thepromoter and terminator in the plasmid can be that of a P. methanolicagene, such as a P. methanolica alcohol utilization gene (AUG1 or AUG2).Other useful promoters include those of the dihydroxyacetone synthase(DHAS), formate dehydrogenase (FMD), and catalase (CAT) genes. Tofacilitate integration of the DNA into the host chromosome, the entireexpression segment of the plasmid can be flanked at both ends by hostDNA sequences. A suitable selectable marker for use in Pichiamethanolica is a P. methanolica ADE2 gene, which encodesphosphoribosyl-5-aminoimidazole carboxylase (AIRC; EC 4.1.1.21), andwhich allows ade2 host cells to grow in the absence of adenine. Forlarge-scale, industrial processes where it is desirable to minimize theuse of methanol, host cells can be used in which both methanolutilization genes (AUG1 and AUG2) are deleted. For production ofsecreted proteins, host cells can be deficient in vacuolar proteasegenes (PEP4 and PRB1). Electroporation is used to facilitate theintroduction of a plasmid containing DNA encoding a polypeptide ofinterest into P. methanolica cells. P. methanolica cells can betransformed by electroporation using an exponentially decaying, pulsedelectric field having a field strength of from 2.5 to 4.5 kV/cm,preferably about 3.75 kV/cm, and a time constant (t) of from 1 to 40milliseconds, most preferably about 20 milliseconds.

[0167] Expression vectors can also be introduced into plant protoplasts,intact plant tissues, or isolated plant cells. Methods for introducingexpression vectors into plant tissue include the direct infection orco-cultivation of plant tissue with Agrobacterium tumefaciens,microprojectile-mediated delivery, DNA injection, electroporation, andthe like. See, for example, Horsch et al., Science 227:1229 (1985),Klein et al., Biotechnology 10:268 (1992), and Miki et al., “Proceduresfor Introducing Foreign DNA into Plants,” in Methods in Plant MolecularBiology and Biotechnology, Glick et al. (eds.), pages 67-88 (CRC Press,1993).

[0168] Alternatively, Zcytor 18 genes can be expressed in prokaryotichost cells. Suitable promoters that can be used to express Zcytor 18polypeptides in a prokaryotic host are well-known to those of skill inthe art and include promoters capable of recognizing the T4, T3, Sp6 andT7 polymerases, the PR and PL promoters of bacteriophage lambda, thetrp, recA, heat shock, lacUV5, tac, Ipp-lacSpr, phoA, and lacZ promotersof E. coli, promoters of B. subtilis, the promoters of thebacteriophages of Bacillus, Streptomyces promoters, the int promoter ofbacteriophage lambda, the bla promoter of pBR322, and the CAT promoterof the chloramphenicol acetyl transferase gene. Prokaryotic promotershave been reviewed by Glick, J. Ind. Microbiol. 1:277 (1987), Watson etal., Molecular Biology of the Gene, 4th Ed. (Benjamin Cummins 1987), andby Ausubel et al. (1995).

[0169] Suitable prokaryotic hosts include E. coli and Bacillus subtilus.Suitable strains of E. coli include BL21(DE3), BL21(DE3)pLysS,BL21(DE3)pLysE, DH1, DH4I, DH5, DH5I, DH51F′, DH51MCR, DH10B, DH10B/p3,DH11S, C600, HB101, JM101, JM105, JM109, JM110, K38, RR1, Y1088, Y1089,CSH18, ER1451, and ER1647 (see, for example, Brown (ed.), MolecularBiology Labfax (Academic Press 1991)). Suitable strains of Bacillussubtilus include BR151, YB886, MI119, MI120, and B170 (see, for example,Hardy, “Bacillus Cloning Methods,” in DNA Cloning: A Practical Approach,Glover (ed.) (IRL Press 1985)).

[0170] When expressing a Zcytor 18 polypeptide in bacteria such as E.coli, the polypeptide may be retained in the cytoplasm, typically asinsoluble granules, or may be directed to the periplasmic space by abacterial secretion sequence. In the former case, the cells are lysed,and the granules are recovered and denatured using, for example,guanidine isothiocyanate or urea. The denatured polypeptide can then berefolded and dimerized by diluting the denaturant, such as by dialysisagainst a solution of urea and a combination of reduced and oxidizedglutathione, followed by dialysis against a buffered saline solution. Inthe latter case, the polypeptide can be recovered from the periplasmicspace in a soluble and functional form by disrupting the cells (by, forexample, sonication or osmotic shock) to release the contents of theperiplasmic space and recovering the protein, thereby obviating the needfor denaturation and refolding.

[0171] Methods for expressing proteins in prokaryotic hosts arewell-known to those of skill in the art (see, for example, Williams etal., “Expression of foreign proteins in E. coli using plasmid vectorsand purification of specific polyclonal antibodies,” in DNA Cloning 2:Expression Systems, 2nd Edition, Glover et al. (eds.), page 15 (OxfordUniversity Press 1995), Ward et al., “Genetic Manipulation andExpression of Antibodies,” in Monoclonal Antibodies: Principles andApplications, page 137 (Wiley-Liss, Inc. 1995), and Georgiou,“Expression of Proteins in Bacteria,” in Protein Engineering: Principlesand Practice, Cleland et al. (eds.), page 101 (John Wiley & Sons, Inc.1996)).

[0172] Standard methods for introducing expression vectors intobacterial, yeast, insect, and plant cells are provided, for example, byAusubel (1995).

[0173] General methods for expressing and recovering foreign proteinproduced by a mammalian cell system are provided by, for example,Etcheverry, “Expression of Engineered Proteins in Mammalian CellCulture,” in Protein Engineering: Principles and Practice, Cleland etal. (eds.), pages 163 (Wiley-Liss, Inc. 1996). Standard techniques forrecovering protein produced by a bacterial system is provided by, forexample, Grisshammer et al., “Purification of over-produced proteinsfrom E. coli cells,” in DNA Cloning 2: Expression Systems, 2nd Edition,Glover et al. (eds.), pages 59-92 (Oxford University Press 1995).Established methods for isolating recombinant proteins from abaculovirus system are described by Richardson (ed.), BaculovirusExpression Protocols (The Humana Press, Inc. 1995).

[0174] As an alternative, polypeptides of the present invention can besynthesized by exclusive solid phase synthesis, partial solid phasemethods, fragment condensation or classical solution synthesis. Thesesynthesis methods are well-known to those of skill in the art (see, forexample, Merrifield, J. Am. Chem. Soc. 85:2149 (1963), Stewart et al.,“Solid Phase Peptide Synthesis” (2nd Edition), (Pierce Chemical Co.1984), Bayer and Rapp, Chem. Pept. Prot. 3:3 (1986), Atherton et al.,Solid Phase Peptide Synthesis: A Practical Approach (IRL Press 1989),Fields and Colowick, “Solid-Phase Peptide Synthesis,” Methods inEnzymology Volume 289 (Academic Press 1997), and Lloyd-Williams et al.,Chemical Approaches to the Synthesis of Peptides and Proteins (CRCPress, Inc. 1997)). Variations in total chemical synthesis strategies,such as “native chemical ligation” and “expressed protein ligation” arealso standard (see, for example, Dawson et al., Science 266:776 (1994),Hackeng et al., Proc. Nat'l Acad. Sci. USA 94:7845 (1997), Dawson,Methods Enzymol. 287: 34 (1997), Muir et al, Proc. Nat'l Acad. Sci. USA95:6705 (1998), and Severinov and Muir, J. Biol. Chem. 273:16205(1998)).

[0175] Peptides and polypeptides of the present invention comprise atleast six, at least nine, or at least 15 contiguous amino acid residuesof SEQ ID NO:2, SEQ ID NO:5, or SEQ ID NO:8. As an illustration,polypeptides can comprise at least six, at least nine, or at least 15contiguous amino acid residues of any of the following amino acidsequences: (a) amino acid residues 1 to 203 of SEQ ID NO:2, (b) aminoacid residues 36 to 203 of SEQ ID NO:2, (c) amino acid residues 36 to313 of SEQ ID NO:2, (d) amino acid residues 1 to 753 of SEQ ID NO:2, (e)amino acid residues 1 to 189 of SEQ ID NO:8, (f) amino acid residues 36to 189 of SEQ ID NO:8, (g) amino acid residues 36 to 299 of SEQ ID NO:8,and (h) amino acid residues 1 to 739 of SEQ 11) NO:8. Within certainembodiments of the invention, the polypeptides comprise 20, 30, 40, 50,100, or more contiguous residues of these amino acid sequences. Forexample, polypeptides can comprise at least 30 contiguous amino acidresidues of an amino acid sequence selected from the group consistingof: (a) amino acid residues 1 to 218 of SEQ ID NO:2, (b) amino acidresidues 36 to 218 of SEQ ID NO:2, (c) amino acid residues 36 to 313 ofSEQ ID NO:2, (d) amino acid residues 1 to 753 of SEQ ID NO:2, (e) aminoacid residues 1 to 204 of SEQ ID NO:8, (f) amino acid residues 36 to 204of SEQ ID NO:8, (g) amino acid residues 36 to 299 of SEQ ID NO:8, and(h) amino acid residues 1 to 739 of SEQ ID NO:8. Nucleic acid moleculesencoding such peptides and polypeptides are useful as polymerase chainreaction primers and probes.

[0176] 6. Production of Zcytor 18 Fusion Proteins and Conjugates

[0177] One general class of Zcytor 18 analogs are variants having anamino acid sequence that is a mutation of the amino acid sequencedisclosed herein. Another general class of Zcytor 18 analogs is providedby anti-idiotype antibodies, and fragments thereof, as described below.Moreover, recombinant antibodies comprising anti-idiotype variabledomains can be used as analogs (see, for example, Monfardini et al.,Proc. Assoc. Am. Physicians 108:420 (1996)). Since the variable domainsof anti-idiotype Zcytor 18 antibodies mimic Zcytor 18, these domains canprovide Zcytor 18 binding activity. Methods of producing anti-idiotypiccatalytic antibodies are known to those of skill in the art (see, forexample, Joron et al., Ann. N Y Acad. Sci. 672:216 (1992), Friboulet etal., Appl. Biocheni. Biotechnol. 47:229 (1994), and Avalle et al., Ann.N YAcad. Sci. 864:118 (1998)).

[0178] Another approach to identifying Zcytor 18 analogs is provided bythe use of combinatorial libraries. Methods for constructing andscreening phage display and other combinatorial libraries are provided,for example, by Kay et al., Phage Display of Peptides and Proteins(Academic Press 1996), Verdine, U.S. Pat. No. 5,783,384, Kay, et. al.,U.S. Pat. No. 5,747,334, and Kauffman et al., U.S. Pat. No. 5,723,323.

[0179] Zcytor 18 polypeptides have both in vivo and in vitro uses. As anillustration, a soluble form of Zcytor 18 can be added to cell culturemedium to inhibit the effects of the Zcytor 18 ligand produced by thecultured cells.

[0180] Fusion proteins of Zcytor 18 can be used to express Zcytor 18 ina recombinant host, and to isolate the produced Zcytor 18. As describedbelow, particular Zcytor 18 fusion proteins also have uses in diagnosisand therapy. One type of fusion protein comprises a peptide that guidesa Zcytor 18 polypeptide from a recombinant host cell. To direct a Zcytor18 polypeptide into the secretory pathway of a eukaryotic host cell, asecretory signal sequence (also known as a signal peptide, a leadersequence, prepro sequence or pre sequence) is provided in the Zcytor 18expression vector. While the secretory signal sequence may be derivedfrom Zcytor 18, a suitable signal sequence may also be derived fromanother secreted protein or synthesized de novo. The secretory signalsequence is operably linked to a Zcytor 18-encoding sequence such thatthe two sequences are joined in the correct reading frame and positionedto direct the newly synthesized polypeptide into the secretory pathwayof the host cell. Secretory signal sequences are commonly positioned 5′to the nucleotide sequence encoding the polypeptide of interest,although certain secretory signal sequences may be positioned elsewherein the nucleotide sequence of interest (see, e.g., Welch et al., U.S.Pat. No. 5,037,743; Holland et al., U.S. Pat. No. 5,143,830).

[0181] Although the secretory signal sequence of Zcytor 18 or anotherprotein produced by mammalian cells (e.g., tissue-type plasminogenactivator signal sequence, as described, for example, in U.S. Pat. No.5,641,655) is useful for expression of Zcytor 18 in recombinantmammalian hosts, a yeast signal sequence is preferred for expression inyeast cells. Examples of suitable yeast signal sequences are thosederived from yeast mating phermone α-factor (encoded by the MFα1 gene),invertase (encoded by the SUC2 gene), or acid phosphatase (encoded bythe PH05 gene). See, for example, Romanos et al., “Expression of ClonedGenes in Yeast,” in DNA Cloning 2: A Practical Approach, 2nd Edition,Glover and Hames (eds.), pages 123-167 (Oxford University Press 1995).

[0182] In bacterial cells, it is often desirable to express aheterologous protein as a fusion protein to decrease toxicity, increasestability, and to enhance recovery of the expressed protein. Forexample, Zcytor 18 can be expressed as a fusion protein comprising aglutathione S-transferase polypeptide. Glutathione S-transferease fusionproteins are typically soluble, and easily purifiable from E. colilysates on immobilized glutathione columns. In similar approaches, aZcytor 18 fusion protein comprising a maltose binding proteinpolypeptide can be isolated with an amylose resin column, while a fusionprotein comprising the C-terminal end of a truncated Protein A gene canbe purified using IgG-Sepharose. Established techniques for expressing aheterologous polypeptide as a fusion protein in a bacterial cell aredescribed, for example, by Williams et al., “Expression of ForeignProteins in E. coli Using Plasmid Vectors and Purification of SpecificPolyclonal Antibodies,” in DNA Cloning 2: A Practical Approach, 2ndEdition, Glover and Hames (Eds.), pages 15-58 (Oxford University Press1995). In addition, commercially available expression systems areavailable. For example, the PINPOINT Xa protein purification system(Promega Corporation; Madison, Wis.) provides a method for isolating afusion protein comprising a polypeptide that becomes biotinylated duringexpression with a resin that comprises avidin.

[0183] Peptide tags that are useful for isolating heterologouspolypeptides expressed by either prokaryotic or eukaryotic cells includepolyHistidine tags (which have an affinity for nickel-chelating resin),c-myc tags, calmodulin binding protein (isolated with calmodulinaffinity chromatography), substance P, the RYIRS tag (which binds withanti-RYIRS antibodies), the Glu-Glu tag, and the FLAG tag (which bindswith anti-FLAG antibodies). See, for example, Luo et al., Arch. Biochem.Biophys. 329:215 (1996), Morganti et al., Biotechnol. Appl. Biochem.23:67 (1996), and Zheng et al., Gene 186:55 (1997). Nucleic acidmolecules encoding such peptide tags are available, for example, fromSigma-Aldrich Corporation (St. Louis, Mo.).

[0184] The present invention also contemplates that the use of thesecretory signal sequence contained in the Zcytor 18 polypeptides of thepresent invention to direct other polypeptides into the secretorypathway. A signal fusion polypeptide can be made wherein a secretorysignal sequence derived from amino acid residues 1 to 35 of SEQ ID NO:2is operably linked to another polypeptide using methods known in the artand disclosed herein. The secretory signal sequence contained in thefusion polypeptides of the present invention is preferably fusedamino-terminally to an additional peptide to direct the additionalpeptide into the secretory pathway. Such constructs have numerousapplications known in the art. For example, these novel secretory signalsequence fusion constructs can direct the secretion of an activecomponent of a normally non-secreted protein, such as a receptor. Suchfusions may be used in a transgenic animal or in a cultured recombinanthost to direct peptides through the secretory pathway. With regard tothe latter, exemplary polypeptides include pharmaceutically activemolecules such as Factor VIIa, proinsulin, insulin, follicle stimulatinghormone, tissue type plasminogen activator, tumor necrosis factor,interleukins (e.g., interleukin-1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16,IL-17, IL-18, IL-19, IL-20, and IL-21), colony stimulating factors(e.g., granulocyte-colony stimulating factor (G-CSF) and granulocytemacrophage-colony stimulating factor (GM-CSF)), interferons (e.g.,interferons-α, -β, -γ, -ω, -δ, -ε, and -τ), the stem cell growth factordesignated “S1 factor,” erythropoietin, and thrombopoietin. The Zcytor18 secretory signal sequence contained in the fusion polypeptides of thepresent invention is preferably fused amino-terminally to an additionalpeptide to direct the additional peptide into the secretory pathway.Fusion proteins comprising a Zcytor 18 secretory signal sequence can beconstructed using standard techniques.

[0185] Another form of fusion protein comprises a Zcytor 18 polypeptideand an immunoglobulin heavy chain constant region, typically an Fcfragment, which contains two or three constant region domains and ahinge region but lacks the variable region. As an illustration, Chang etal., U.S. Pat. No. 5,723,125, describe a fusion protein comprising ahuman interferon and a human immunoglobulin Fc fragment. The C-terminalof the interferon is linked to the N-terminal of the Fc fragment by apeptide linker moiety. An example of a peptide linker is a peptidecomprising primarily a T cell inert sequence, which is immunologicallyinert. An exemplary peptide linker has the amino acid sequence: GGSGGSGGGG SGGGG S (SEQ ID NO:10.). In this fusion protein, an illustrativeFc moiety is a human γ4 chain, which is stable in solution and haslittle or no complement activating activity. Accordingly, the presentinvention contemplates a Zcytor 18 fusion protein that comprises aZcytor 18 moiety and a human Fc fragment, wherein the C-terminus of theZcytor 18 moiety is attached to the N-terminus of the Fc fragment via apeptide linker, such as a peptide consisting of the amino acid sequenceof SEQ ID NO:10. The Zcytor 18 moiety can be a Zcytor 18 molecule or afragment thereof. For example, a fusion protein can comprise an Fcfragment (e.g., a human Fc fragment), and amino acid residues 36 to 313of SEQ ID NO:2, amino acid residues 36 to 189 of SEQ ID NO:2, amino acidresidues 36 to 299 of SEQ ID NO:8, or amino acid residues 36 to 175 ofSEQ ID NO:8.

[0186] In another variation, a Zcytor 18 fusion protein comprises an IgGsequence, a Zcytor 18 moiety covalently joined to the aminoterminal endof the IgG sequence, and a signal peptide that is covalently joined tothe aminoterminal of the Zcytor 18 moiety, wherein the IgG sequenceconsists of the following elements in the following order: a hingeregion, a CH₂ domain, and a CH₃ domain. Accordingly, the IgG sequencelacks a CH₁ domain. The Zcytor 18 moiety displays a Zcytor 18 activity,as described herein, such as the ability to bind with a Zcytor 18ligand. This general approach to producing fusion proteins that compriseboth antibody and nonantibody portions has been described by LaRochelleet al., EP 742830 (WO 95/21258).

[0187] Fusion proteins comprising a Zcytor 18 moiety and an Fc moietycan be used, for example, as an in vitro assay tool. For example, thepresence of a Zcytor 18 ligand in a biological sample can be detectedusing a Zcytor 18-immunoglobulin fusion protein, in which the Zcytor 18moiety is used to bind the ligand, and a macromolecule, such as ProteinA or anti-Fc antibody, is used to bind the fusion protein to a solidsupport. Such systems can be used to identify agonists and antagoniststhat interfere with the binding of a Zcytor 18 ligand to its receptor.

[0188] Other examples of antibody fusion proteins include polypeptidesthat comprise an antigen-binding domain and a Zcytor 18 fragment thatcontains a Zcytor 18 extracellular domain. Such molecules can be used totarget particular tissues for the benefit of Zcytor 18 binding activity.

[0189] The present invention further provides a variety of otherpolypeptide fusions. For example, part or all of a domain(s) conferringa biological function can be swapped between Zcytor 18 of the presentinvention with the functionally equivalent domain(s) from another memberof the cytokine receptor family. Polypeptide fusions can be expressed inrecombinant host cells to produce a variety of Zcytor 18 fusion analogs.A Zcytor 18 polypeptide can be fused to two or more moieties or domains,such as an affinity tag for purification and a targeting domain.Polypeptide fusions can also comprise one or more cleavage sites,particularly between domains. See, for example, Tuan et al., ConnectiveTissue Research 34:1 (1996)., Fusion proteins can be prepared by methodsknown to those skilled in the art by preparing each component of thefusion protein and chemically conjugating them. Alternatively, apolynucleotide encoding both components of the fusion protein in theproper reading frame can be generated using known techniques andexpressed by the methods described herein. General methods for enzymaticand chemical cleavage of fusion proteins are described, for example, byAusubel (1995) at pages 16-19 to 1625.

[0190] Zcytor 18 polypeptides can be used to identify and to isolateZcytor 18 ligands. For example, proteins and peptides of the presentinvention can be immobilized on a column and used to bind ligands from abiological sample that is run over the column (Hermanson et al. (eds.),Immobilized Affinity Ligand Techniques, pages 195-202 (Academic Press1992)).

[0191] The activity of a Zcytor 18 polypeptide can be observed by asilicon-based biosensor microphysiometer, which measures theextracellular acidification rate or proton excretion associated withreceptor binding and subsequent physiologic cellular responses. Anexemplary device is the CYTOSENSOR Microphysiometer manufactured byMolecular Devices, Sunnyvale, Calif. A variety of cellular responses,such as cell proliferation, ion transport, energy production,inflammatory response, regulatory and receptor activation, and the like,can be measured by this method (see, for example, McConnell et al.,Science 257:1906 (1992), Pitchford et al., Meth. Enzymol. 228:84 (1997),Arimilli et al., J. Immunol. Meth. 212:49 (1998), Van Liefde et al.,Eur. J. Pharmacol. 346:87 (1998)). The microphysiometer can be used forassaying eukaryotic, prokaryotic, adherent, or non-adherent cells. Bymeasuring extracellular acidification changes in cell media over time,the microphysiometer directly measures cellular responses to variousstimuli, including agonists, ligands, or antagonists of Zcytor 18.

[0192] For example, the microphysiometer is used to measure responses ofan Zcytor 18-expressing eukaryotic cell, compared to a controleukaryotic cell that does not express Zcytor 18 polypeptide. Suitablecells responsive to Zcytor 18-modulating stimuli include recombinanthost cells comprising a Zcytor 18 expression vector, and cells thatnaturally express Zcytor 18. Extracellular acidification provides onemeasure for a Zcytor 18-modulated cellular response. In addition, thisapproach can be used to identify ligands, agonists, and antagonists ofZcytor 18 ligand. For example, a molecule can be identified as anagonist of Zcytor 18 ligand by providing cells that express a Zcytor 18polypeptide, culturing a first portion of the cells in the absence ofthe test compound, culturing a second portion of the cells in thepresence of the test compound, and determining whether the secondportion exhibits a cellular response, in comparison with the firstportion.

[0193] Alternatively, a solid phase system can be used to identify aZcytor 18 ligand, or an agonist or antagonist of a Zcytor 18 ligand. Forexample, a Zcytor 18 polypeptide or Zcytor 18 fusion protein can beimmobilized onto the surface of a receptor chip of a commerciallyavailable biosensor instrument (BIACORE, Biacore AB; Uppsala, Sweden).The use of this instrument is disclosed, for example, by Karlsson,Immunol. Methods 145:229 (1991), and Cunningham and Wells, J. Mol. Biol.234:554 (1993).

[0194] In brief, a Zcytor 18 polypeptide or fusion protein is covalentlyattached, using amine or sulfhydryl chemistry, to dextran fibers thatare attached to gold film within a flow cell. A test sample is thenpassed through the cell. If a ligand is present in the sample, it willbind to the immobilized polypeptide or fusion protein, causing a changein the refractive index of the medium, which is detected as a change insurface plasmon resonance of the gold film. This system allows thedetermination of on- and off-rates, from which binding affinity can becalculated, and assessment of stoichiometry of binding. This system canalso be used to examine antibody-antigen interactions, and theinteractions of other complement/anti-complement pairs.

[0195] Zcytor 18 binding domains can be further characterized byphysical analysis of structure, as determined by such techniques asnuclear magnetic resonance, crystallography, electron diffraction orphotoaffinity labeling, in conjunction with mutation of putative contactsite amino acids of Zcytor 18 ligand agonists. See, for example, de Voset al., Science 255:306 (1992), Smith et al., J. Mol. Biol. 224:899(1992), and Wlodaver et al., FEBS Lett. 309:59 (1992).

[0196] The present invention also contemplates chemically modifiedZcytor 18 compositions, in which a Zcytor 18 polypeptide is linked witha polymer. Illustrative Zcytor 18 polypeptides are soluble polypeptidesthat lack a functional transmembrane domain, such as a polypeptideconsisting of amino acid residues 36 to 313 of SEQ ID NO:2, amino acidresidues 36 to 189 of SEQ ID NO:2, amino acid residues 36 to 299 of SEQID NO:8, or amino acid residues 36 to 175 of SEQ ID NO:8. Typically, thepolymer is water-soluble so that the Zcytor 18 conjugate does notprecipitate in an aqueous environment, such as a physiologicalenvironment. An example of a suitable polymer is one that has beenmodified to have a single reactive group, such as an active ester foracylation, or an aldehyde for alkylation, In this way, the degree ofpolymerization can be controlled. An example of a reactive aldehyde ispolyethylene glycol propionaldehyde, or mono-(C1-C10) alkoxy, or aryloxyderivatives thereof (see, for example, Harris, et al., U.S. Pat. No.5,252,714). The polymer may be branched or unbranched. Moreover, amixture of polymers can be used to produce Zcytor 18 conjugates.

[0197] Zcytor 18 conjugates used for therapy can comprisepharmaceutically acceptable water-soluble polymer moieties. Suitablewater-soluble polymers include polyethylene glycol (PEG),monomethoxy-PEG, mono-(C1-C10)alkoxy-PEG, aryloxyPEG, poly-(N-vinylpyrrolidone)PEG, tresyl monomethoxy PEG, PEG propionaldehyde,bis-succinimidyl carbonate PEG, propylene glycol homopolymers, apolypropylene oxide/ethylene oxide co-polymer, polyoxyethylated polyols(e.g., glycerol), polyvinyl alcohol, dextran, cellulose, or othercarbohydrate-based polymers. Suitable PEG may have a molecular weightfrom about 600 to about 60,000, including, for example, 5,000, 12,000,20,000 and 25,000. A Zcytor 18 conjugate can also comprise a mixture ofsuch water-soluble polymers.

[0198] One example of a Zcytor 18 conjugate comprises a Zcytor 18 moietyand a polyalkyl oxide moiety attached to the N-terminus of the Zcytor 18moiety. PEG is one suitable polyalkyl oxide. As an illustration, Zcytor18 can be modified with PEG, a process known as “PEGylation.” PEGylationof Zcytor 18 can be carried out by any of the PEGylation reactions knownin the art (see, for example, EP 0 154 316, Delgado et al., CriticalReviews in Therapeutic Drug Carrier Systems 9:249 (1992), Duncan andSpreafico, Clin. Pharmacokinet. 27:290 (1994), and Francis et al., Int JHematol 68:1 (1998)). For example, PEGylation can be performed by anacylation reaction or by an alkylation reaction with a reactivepolyethylene glycol molecule. In an alternative approach, Zcytor 18conjugates are formed by condensing activated PEG, in which a terminalhydroxy or amino group of PEG has been replaced by an activated linker(see, for example, Karasiewicz et al., U.S. Pat. No. 5,382,657).

[0199] PEGylation by acylation typically requires reacting an activeester derivative of PEG with a Zcytor 18 polypeptide. An example of anactivated PEG ester is PEG esterified to N-hydroxysuccinimide. As usedherein, the term “acylation” includes the following types of linkagesbetween Zcytor 18 and a water-soluble polymer: amide, carbamate,urethane, and the like. Methods for preparing PEGylated Zcytor 18 byacylation will typically comprise the steps of (a) reacting a Zcytor 18polypeptide with PEG (such as a reactive ester of an aldehyde derivativeof PEG) under conditions whereby one or more PEG groups attach to Zcytor18, and (b) obtaining the reaction product(s). Generally, the optimalreaction conditions for acylation reactions will be determined basedupon known parameters and desired results. For example, the larger theratio of PEG:Zcytor 18, the greater the percentage of polyPEGylatedZcytor 18 product.

[0200] The product of PEGylation by acylation is typically apolyPEGylated Zcytor 18 product, wherein the lysine ε-amino groups arePEGylated via an acyl linking group. An example of a connecting linkageis an amide. Typically, the resulting Zcytor 18 will be at least 95%mono-, di-, or tri-pegylated, although some species with higher degreesof PEGylation may be formed depending upon the reaction conditions.PEGylated species can be separated from unconjugated Zcytor 18polypeptides using standard purification methods, such as dialysis,ultrafiltration, ion exchange chromatography, affinity chromatography,and the like.

[0201] PEGylation by alkylation generally involves reacting a terminalaldehyde derivative of PEG with Zcytor 18 in the presence of a reducingagent. PEG groups can be attached to the polypeptide via a —CH₂—NHgroup.

[0202] Derivatization via reductive alkylation to produce amonoPEGylated product takes advantage of the differential reactivity ofdifferent types of primary amino groups available for derivatization.Typically, the reaction is performed at a pH that allows one to takeadvantage of the pKa differences between the ε-amino groups of thelysine residues and the α-amino group of the N-terminal residue of theprotein. By such selective derivatization, attachment of a water-solublepolymer that contains a reactive group such as an aldehyde, to a proteinis controlled. The conjugation with the polymer occurs predominantly atthe N-terminus of the protein without significant modification of otherreactive groups such as the lysine side chain amino groups. The presentinvention provides a substantially homogenous preparation of Zcytor 18monopolymer conjugates.

[0203] Reductive alkylation to produce a substantially homogenouspopulation of monopolymer Zcytor 18 conjugate molecule can comprise thesteps of: (a) reacting a Zcytor 18 polypeptide with a reactive PEG underreductive alkylation conditions at a pH suitable to permit selectivemodification of the α-amino group at the amino terminus of the Zcytor18, and (b) obtaining the reaction product(s). The reducing agent usedfor reductive alkylation should be stable in aqueous solution and ableto reduce only the Schiff base formed in the initial process ofreductive alkylation. Illustrative reducing agents include sodiumborohydride, sodium cyanoborohydride, dimethylamine borane,trimethylamine borane, and pyridine borane.

[0204] For a substantially homogenous population of monopolymer Zcytor18 conjugates, the reductive alkylation reaction conditions are thosewhich permit the selective attachment of the water soluble polymermoiety to the N-terminus of Zcytor 18. Such reaction conditionsgenerally provide for pKa differences between the lysine amino groupsand the α-amino group at the N-terminus. The pH also affects the ratioof polymer to protein to be used. In general, if the pH is lower, alarger excess of polymer to protein will be desired because the lessreactive the N-terminal α-group, the more polymer is needed to achieveoptimal conditions. If the pH is higher, the polymer:Zcytor 18 need notbe as large because more reactive groups are available. Typically, thepH will fall within the range of 3 to 9, or 3 to 6.

[0205] Another factor to consider is the molecular weight of thewater-soluble polymer. Generally, the higher the molecular weight of thepolymer, the fewer number of polymer molecules which may be attached tothe protein. For PEGylation reactions, the typical molecular weight isabout 2 kDa to about 100 kDa, about 5 kDa to about 50 kDa, or about 12kDa to about 25 kDa. The molar ratio of water-soluble polymer to Zcytor18 will generally be in the range of 1:1 to 100:1. Typically, the molarratio of water-soluble polymer to Zcytor 18 will be 1:1 to 20:1 forpolyPEGylation, and 1:1 to 5:1 for monoPEGylation.

[0206] General methods for producing conjugates comprising a polypeptideand water-soluble polymer moieties are known in the art. See, forexample, Karasiewicz et al., U.S. Pat. No. 5,382,657, Greenwald et al.,U.S. Pat. No. 5,738,846, Nieforth et al., Clin. Pharmacol. Ther. 59:636(1996), Monkarsh et al., Anal. Biochem. 247:434 (1997)).

[0207] The present invention contemplates compositions comprising apeptide or polypeptide described herein. Such compositions can furthercomprise a carrier. The carrier can be a conventional organic orinorganic carrier. Examples of carriers include water, buffer solution,alcohol, propylene glycol, macrogol, sesame oil, corn oil, and the like.

[0208] 7. Isolation of Zcytor 18 Polypeptides

[0209] The polypeptides of the present invention can be purified to atleast about 80% purity, to at least about 90% purity, to at least about95% purity, or greater than 95% purity with respect to contaminatingmacromolecules, particularly other proteins and nucleic acids, and freeof infectious and pyrogenic agents. The polypeptides of the presentinvention may also be purified to a pharmaceutically pure state, whichis greater than 99.9% pure. In certain preparations, purifiedpolypeptide is substantially free of other polypeptides, particularlyother polypeptides of animal origin.

[0210] Fractionation and/or conventional purification methods can beused to obtain preparations of Zcytor 18 purified from natural sources(e.g., testis tissue), synthetic Zcytor 18 polypeptides, and recombinantZcytor 18 polypeptides and fusion Zcytor 18 polypeptides purified fromrecombinant host cells. In general, ammonium sulfate precipitation andacid or chaotrope extraction may be used for fractionation of samples.Exemplary purification steps may include hydroxyapatite, size exclusion,FPLC and reverse-phase high performance liquid chromatography. Suitablechromatographic media include derivatized dextrans, agarose, cellulose,polyacrylamide, specialty silicas, and the like. PEI, DEAE, QAE and Qderivatives are suitable. Exemplary chromatographic media include thosemedia derivatized with phenyl, butyl, or octyl groups, such asPhenyl-Sepharose FF (Pharmacia), Toyopearl butyl 650 (Toso Haas,Montgomeryville, Pa.), Octyl-Sepharose (Pharmacia) and the like; orpolyacrylic resins, such as Amberchrom CG 71 (Toso Haas) and the like.Suitable solid supports include glass beads, silica-based resins,cellulosic resins, agarose beads, cross-linked agarose beads,polystyrene beads, cross-linked polyacrylamide resins and the like thatare insoluble under the conditions in which they are to be used. Thesesupports may be modified with reactive groups that allow attachment ofproteins by amino groups, carboxyl groups, sulfhydryl groups, hydroxylgroups and/or carbohydrate moieties.

[0211] Examples of coupling chemistries include cyanogen bromideactivation, N-hydroxysuccinimide activation, epoxide activation,sulfhydryl activation, hydrazide activation, and carboxyl and aminoderivatives for carbodiimide coupling chemistries. These and other solidmedia are well known and widely used in the art, and are available fromcommercial suppliers. Selection of a particular method for polypeptideisolation and purification is a matter of routine design and isdetermined in part by the properties of the chosen support. See, forexample, Affinity Chromatography: Principles & Methods (Pharmacia LKBBiotechnology 1988), and Doonan, Protein Purification Protocols (TheHumana Press 1996).

[0212] Additional variations in Zcytor 18 isolation and purification canbe devised by those of skill in the art. For example, anti-Zcytor 18antibodies, obtained as described below, can be used to isolate largequantities of protein by immunoaffinity purification.

[0213] The polypeptides of the present invention can also be isolated byexploitation of particular properties. For example, immobilized metalion adsorption (IMAC) chromatography can be used to purifyhistidine-rich proteins, including those comprising polyhistidine tags.Briefly, a gel is first charged with divalent metal ions to form achelate (Sulkowski, Trends in Biochem. 3:1 (1985)). Histidine-richproteins will be adsorbed to this matrix with differing affinities,depending upon the metal ion used, and will be eluted by competitiveelution, lowering the pH, or use of strong chelating agents. Othermethods of purification include purification of glycosylated proteins bylectin affinity chromatography and ion exchange chromatography (M.Deutscher, (ed.), Meth. Enzymol. 182:529 (1990)). Within additionalembodiments of the invention, a fusion of the polypeptide of interestand an affinity tag (e.g., maltose-binding protein, an immunoglobulindomain) may be constructed to facilitate purification.

[0214] Zcytor 18 polypeptides or fragments thereof may also be preparedthrough chemical synthesis, as described above. Zcytor 18 polypeptidesmay be monomers or multimers; glycosylated or non-glycosylated;PEGylated or non-PEGylated; and may or may not include an initialmethionine amino acid residue.

[0215] 8. Production of Antibodies to Zcytor 18 Proteins

[0216] Antibodies to Zcytor 18 can be obtained, for example, using theproduct of a Zcytor 18 expression vector or Zcytor 18 isolated from anatural source as an antigen. Particularly useful anti-Zcytor 18antibodies “bind specifically” with Zcytor 18. Antibodies are consideredto be specifically binding if the antibodies exhibit at least one of thefollowing two properties: (1) antibodies bind to Zcytor 18 with athreshold level of binding activity, and (2) antibodies do notsignificantly cross-react with polypeptides related to Zcytor 18.

[0217] With regard to the first characteristic, antibodies specificallybind if they bind to a Zcytor 18 polypeptide, peptide or epitope with abinding affinity (Ka) of 10⁶ M⁻¹ or greater, preferably 10⁷ M⁻¹ orgreater, more preferably 108 M-1 or greater, and most preferably 10⁹ M⁻¹or greater. The binding affinity of an antibody can be readilydetermined by one of ordinary skill in the art, for example, byScatchard analysis (Scatchard, Ann. NY Acad. Sci. 51:660 (1949)). Withregard to the second characteristic, antibodies do not significantlycross-react with related polypeptide molecules, for example, if theydetect Zcytor 18, but not presently known polypeptides using a standardWestern blot analysis. Examples of known related polypeptides includeknown cytokine receptors.

[0218] Anti-Zcytor 18 antibodies can be produced using antigenic Zcytor18 epitope-bearing peptides and polypeptides. Antigenic epitope-bearingpeptides and polypeptides of the present invention contain a sequence ofat least nine, or between 15 to about 30 amino acids contained withinSEQ ID NO:2 or another amino acid sequence disclosed herein. However,peptides or polypeptides comprising a larger portion of an amino acidsequence of the invention, containing from 30 to 50 amino acids, or anylength up to and including the entire amino acid sequence of apolypeptide of the invention, also are useful for inducing antibodiesthat bind with Zcytor 18. It is desirable that the amino acid sequenceof the epitope-bearing peptide is selected to provide substantialsolubility in aqueous solvents (i.e., the sequence includes relativelyhydrophilic residues, while hydrophobic residues are typically avoided).Moreover, amino acid sequences containing proline residues may be alsobe desirable for antibody production.

[0219] As an illustration, potential antigenic sites in Zcytor 18 wereidentified using the Jameson-Wolf method, Jameson and Wolf, CABIOS4:181, (1988), as implemented by the PROTEAN program (version 3.14) ofLASERGENE (DNASTAR; Madison, Wis.). Default parameters were used in thisanalysis.

[0220] The Jameson-Wolf method predicts potential antigenic determinantsby combining six major subroutines for protein structural prediction.Briefly, the HoppWoods method, Hopp et al., Proc. Nat'l Acad. Sci. USA78:3824 (1981), was first used to identify amino acid sequencesrepresenting areas of greatest local hydrophilicity (parameter: sevenresidues averaged). In the second step, Emini's method, Emini et al., J.Virology 55:836 (1985), was used to calculate surface probabilities(parameter: surface decision threshold (0.6)=1). Third, theKarplus-Schultz method, Karplus and Schultz, Naturwissenschaften 72:212(1985), was used to predict backbone chain flexibility (parameter:flexibility threshold (0.2)=1). In the fourth and fifth steps of theanalysis, secondary structure predictions were applied to the data usingthe methods of Chou-Fasman, Chou, “Prediction of Protein StructuralClasses from Amino Acid Composition,” in Prediction of Protein Structureand the Principles of Protein Conformation, Fasman (ed.), pages 549-586(Plenum Press 1990), and Gamier-Robson, Gamier et al., J. Mol. Biol.120:97 (1978) (Chou-Fasman parameters: conformation table=64 proteins; αregion threshold=103; β region threshold=105; Gamier-Robson parameters:α and β decision constants=0). In the sixth subroutine, flexibilityparameters and hydropathy/solvent accessibility factors were combined todetermine a surface contour value, designated as the “antigenic index.”Finally, a peak broadening function was applied to the antigenic index,which broadens major surface peaks by adding 20, 40, 60, or 80% of therespective peak value to account for additional free energy derived fromthe mobility of surface regions relative to interior regions. Thiscalculation was not applied, however, to any major peak that resides ina helical region, since helical regions tend to be less flexible.

[0221] The results of this analysis indicated that the following aminoacid sequences of SEQ ID NO:2 would provide suitable antigenicmolecules: amino acids 29 to 41, amino acids 56 to 67, 131 to 139, aminoacids 145 to 161, amino acids 214 to 227, amino acids 251 to 271, aminoacids 347 to 357, amino acids 362 to 371, amino acids 445 to 462, aminoacids 526 to 555, amino acids 628 to 654, amino acids 665 to 688, andamino acids 695 to 735, while amino acids 45 to 53 of SEQ ID NO:8 wouldprovide a suitable antigenic molecule. The present inventioncontemplates the use of any one of these antigenic amino acid sequencesto generate antibodies to Zcytor 18. The present invention alsocontemplates polypeptides comprising at least one of these antigenicmolecules.

[0222] Polyclonal antibodies to recombinant Zcytor 18 protein or toZcytor 18 isolated from natural sources can be prepared using methodswell-known to those of skill in the art. See, for example, Green et al.,“Production of Polyclonal Antisera,” in Immunochemical Protocols(Manson, ed.), pages 1-5 (Humana Press 1992), and Williams et al.,“Expression of foreign proteins in E. coli using plasmid vectors andpurification of specific polyclonal antibodies,” in DNA Cloning 2:Expression Systems, 2nd Edition, Glover et al. (eds.), page 15 (OxfordUniversity Press 1995). The immunogenicity of a Zcytor 18 polypeptidecan be increased through the use of an adjuvant, such as alum (aluminumhydroxide) or Freund's complete or incomplete adjuvant. Polypeptidesuseful for immunization also include fusion polypeptides, such asfusions of Zcytor 18 or a portion thereof with an immunoglobulinpolypeptide or with maltose binding protein. The polypeptide immunogenmay be a full-length molecule or a portion thereof. If the polypeptideportion is “hapten-like,” such portion may be advantageously joined orlinked to a macromolecular carrier (such as keyhole limpet hemocyanin(KLH), bovine serum albumin (BSA) or tetanus toxoid) for immunization.

[0223] Although polyclonal antibodies are typically raised in animalssuch as horses, cows, dogs, chicken, rats, mice, rabbits, guinea pigs,goats, or sheep, an anti-Zcytor 18 antibody of the present invention mayalso be derived from a subhuman primate antibody. General techniques forraising diagnostically and therapeutically useful antibodies in baboonsmay be found, for example, in Goldenberg et al., international patentpublication No. WO 91/11465, and in Losman et al., Int. J. Cancer 46:310(1990).

[0224] Alternatively, monoclonal anti-Zcytor 18 antibodies can begenerated. Rodent monoclonal antibodies to specific antigens may beobtained by methods known to those skilled in the art (see, for example,Kohler et al., Nature 256:495 (1975), Coligan et al. (eds.), CurrentProtocols in Immunology, Vol. 1, pages 2.5.1-2.6.7 (John Wiley & Sons1991) [“Coligan”], Picksley et al., “Production of monoclonal antibodiesagainst proteins expressed in E. coli,” in DNA Cloning 2: ExpressionSystems, 2nd Edition, Glover et al. (eds.), page 93 (Oxford UniversityPress 1995)).

[0225] Briefly, monoclonal antibodies can be obtained by injecting micewith a composition comprising a Zcytor 18 gene product, verifying thepresence of antibody production by removing a serum sample, removing thespleen to obtain B-lymphocytes, fusing the B-lymphocytes with myelomacells to produce hybridomas, cloning the hybridomas, selecting positiveclones which produce antibodies to the antigen, culturing the clonesthat produce antibodies to the antigen, and isolating the antibodiesfrom the hybridoma cultures.

[0226] In addition, an anti-Zcytor 18 antibody of the present inventionmay be derived from a human monoclonal antibody. Human monoclonalantibodies are obtained from transgenic mice that have been engineeredto produce specific human antibodies in response to antigenic challenge.In this technique, elements of the human heavy and light chain locus areintroduced into strains of mice derived from embryonic stem cell linesthat contain targeted disruptions of the endogenous heavy chain andlight chain loci. The transgenic mice can synthesize human antibodiesspecific for human antigens, and the mice can be used to produce humanantibody-secreting hybridomas. Methods for obtaining human antibodiesfrom transgenic mice are described, for example, by Green et al., NatureGenet. 7:13 (1994), Lonberg et al., Nature 368:856 (1994), and Taylor etal., Int. Immun. 6:579 (1994).

[0227] Monoclonal antibodies can be isolated and purified from hybridomacultures by a variety of well-established techniques. Such isolationtechniques include affinity chromatography with Protein-A Sepharose,size-exclusion chromatography, and ion-exchange chromatography (see, forexample, Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3; Baines etal., “Purification of Immunoglobulin G (IgG),” in Methods in MolecularBiology, Vol. 10, pages 79-104 (The Humana Press, Inc. 1992)).

[0228] For particular uses, it may be desirable to prepare fragments ofanti-Zcytor 18 antibodies. Such antibody fragments can be obtained, forexample, by proteolytic hydrolysis of the antibody. Antibody fragmentscan be obtained by pepsin or papain digestion of whole antibodies byconventional methods. As an illustration, antibody fragments can beproduced by enzymatic cleavage of antibodies with pepsin to provide a 5Sfragment denoted F(ab′)₂. This fragment can be further cleaved using athiol reducing agent to produce 3.5S Fab′ monovalent fragments.Optionally, the cleavage reaction can be performed using a blockinggroup for the sulfhydryl groups that result from cleavage of disulfidelinkages. As an alternative, an enzymatic cleavage using pepsin producestwo monovalent Fab fragments and an Fc fragment directly. These methodsare described, for example, by Goldenberg, U.S. Pat. No. 4,331,647,Nisonoff et al., Arch Biochem. Biophys. 89:230 (1960), Porter, Biochem.J. 73:119 (1959), Edelman et al., in Methods in Enzymology Vol. 1, page422 (Academic Press 1967), and by Coligan at pages 2.8.1-2.8.10 and2.10.-2.10.4.

[0229] Other methods of cleaving antibodies, such as separation of heavychains to form monovalent light-heavy chain fragments, further cleavageof fragments, or other enzymatic, chemical or genetic techniques mayalso be used, so long as the fragments bind to the antigen that isrecognized by the intact antibody.

[0230] For example, Fv fragments comprise an association of V_(H) andV_(L) chains. This association can be noncovalent, as described by Inbaret al., Proc. Nat'l Acad. Sci. USA 69:2659 (1972). Alternatively, thevariable chains can be linked by an intermolecular disulfide bond orcross-linked by chemicals such as glutaraldehyde (see, for example,Sandhu, Crit. Rev. Biotech. 12:437 (1992)).

[0231] The Fv fragments may comprise V_(H) and V_(L) chains, which areconnected by a peptide linker. These single-chain antigen bindingproteins (scFv) are prepared by constructing a structural genecomprising DNA sequences encoding the V_(H) and V_(L) domains which areconnected by an oligonucleotide. The structural gene is inserted into anexpression vector, which is subsequently introduced into a host cell,such as E. coli. The recombinant host cells synthesize a singlepolypeptide chain with a linker peptide bridging the two V domains.Methods for producing scFvs are described, for example, by Whitlow etal., Methods: A Companion to Methods in Enzymology 2:97 (1991) (alsosee, Bird et al., Science 242:423 (1988), Ladner et al., U.S. Pat. No.4,946,778, Pack et al., Bio/Technology 11:1271 (1993), and Sandhu,supra).

[0232] As an illustration, a scFV can be obtained by exposinglymphocytes to Zcytor 18 polypeptide in vitro, and selecting antibodydisplay libraries in phage or similar vectors (for instance, through useof immobilized or labeled Zcytor 18 protein or peptide). Genes encodingpolypeptides having potential Zcytor 18 polypeptide binding domains canbe obtained by screening random peptide libraries displayed on phage(phage display) or on bacteria, such as E. coli. Nucleotide sequencesencoding the polypeptides can be obtained in a number of ways, such asthrough random mutagenesis and random polynucleotide synthesis. Theserandom peptide display libraries can be used to screen for peptides,which interact with a known target which can be a protein orpolypeptide, such as a ligand or receptor, a biological or syntheticmacromolecule, or organic or inorganic substances. Techniques forcreating and screening such random peptide display libraries are knownin the art (Ladner et al., U.S. Pat. No. 5,223,409, Ladner et al., U.S.Pat. No. 4,946,778, Ladner et al., U.S. Pat. No. 5,403,484, Ladner etal., U.S. Pat. No. 5,571,698, and Kay et al., Phage Display of Peptidesand Proteins (Academic Press, Inc. 1996)) and random peptide displaylibraries and kits for screening such libraries are availablecommercially, for instance from CLONTECH Laboratories, Inc. (Palo Alto,Calif.), Invitrogen Inc. (San Diego, Calif.), New England Biolabs, Inc.(Beverly, Mass.), and Pharmacia LKB Biotechnology Inc. (Piscataway,N.J.). Random peptide display libraries can be screened using the Zcytor18 sequences disclosed herein to identify proteins which bind to Zcytor18.

[0233] Another form of an antibody fragment is a peptide coding for asingle complementarity-determining region (CDR). CDR peptides (“minimalrecognition units”) can be obtained by constructing genes encoding theCDR of an antibody of interest. Such genes are prepared, for example, byusing the polymerase chain reaction to synthesize the variable regionfrom RNA of antibody-producing cells (see, for example, Larrick et al.,Methods: A Companion to Methods in Enzymology 2:106 (1991),Courtenay-Luck, “Genetic Manipulation of Monoclonal Antibodies,” inMonoclonal Antibodies: Production, Engineering and Clinical Application,Ritter et al. (eds.), page 166 (Cambridge University Press 1995), andWard et al., “Genetic Manipulation and Expression of Antibodies,” inMonoclonal Antibodies: Principles and Applications, Birch et al.,(eds.), page 137 (Wiley-Liss, Inc. 1995)).

[0234] Alternatively, an anti-Zcytor 18 antibody may be derived from a“humanized” monoclonal antibody. Humanized monoclonal antibodies areproduced by transferring mouse complementary determining regions fromheavy and light variable chains of the mouse immunoglobulin into a humanvariable domain. Typical residues of human antibodies are thensubstituted in the framework regions of the murine counterparts. The useof antibody components derived from humanized monoclonal antibodiesobviates potential problems associated with the immunogenicity of murineconstant regions. General techniques for cloning murine immunoglobulinvariable domains are described, for example, by Orlandi et al., Proc.Nat'l Acad. Sci. USA 86:3833 (1989). Techniques for producing humanizedmonoclonal antibodies are described, for example, by Jones et al.,Nature 321:522 (1986), Carter et al., Proc. Nat'l Acad. Sci. USA 89:4285(1992), Sandhu, Crit. Rev. Biotech. 12:437 (1992), Singer et al., J.Immun. 150:2844 (1993), Sudhir (ed.), Antibody Engineering Protocols(Humana Press, Inc. 1995), Kelley, “Engineering Therapeutic Antibodies,”in Protein Engineering: Principles and Practice, Cleland et al. (eds.),pages 399-434 (John Wiley & Sons, Inc. 1996), and by Queen et al., U.S.Pat. No. 5,693,762 (1997).

[0235] Polyclonal anti-idiotype antibodies can be prepared by immunizinganimals with anti-Zcytor 18 antibodies or antibody fragments, usingstandard techniques. See, for example, Green et al., “Production ofPolyclonal Antisera,” in Methods In Molecular Biology: ImmunochemicalProtocols, Manson (ed.), pages 1-12 (Humana Press 1992). Also, seeColigan at pages 2.4.1-2.4.7. Alternatively, monoclonal anti-idiotypeantibodies can be prepared using anti-Zcytor 18 antibodies or antibodyfragments as immunogens with the techniques, described above. As anotheralternative, humanized anti-idiotype antibodies or subhuman primateanti-idiotype antibodies can be prepared using the above-describedtechniques. Methods for producing anti-idiotype antibodies aredescribed, for example, by Irie, U.S. Pat. No. 5,208,146, Greene, et.al., U.S. Pat. No. 5,637,677, and Varthakavi and Minocha, J. Gen. Virol.77:1875 (1996).

[0236] 9. Use of Zcytor 18 Nucleotide Sequences to Detect GeneExpression and Gene Structure

[0237] Nucleic acid molecules can be used to detect the expression of aZcytor 18 gene in a biological sample. Suitable probe molecules includedouble-stranded nucleic acid molecules comprising the nucleotidesequence of SEQ ID NO:1, or a portion thereof, as well assingle-stranded nucleic acid molecules having the complement of thenucleotide sequence of SEQ ID NO:1, or a portion thereof. Probemolecules may be DNA, RNA, oligonucleotides, and the like. As usedherein, the term “portion” refers to at least eight nucleotides to atleast 20 or more nucleotides. Illustrative probes bind with regions ofthe Zcytor 18 gene that have a low sequence similarity to comparableregions in other cytokine receptor genes.

[0238] In a basic assay, a single-stranded probe molecule is incubatedwith RNA, isolated from a biological sample, under conditions oftemperature and ionic strength that promote base pairing between theprobe and target Zcytor 18 RNA species. After separating unbound probefrom hybridized molecules, the amount of hybrids is detected.

[0239] Well-established hybridization methods of RNA detection includenorthern analysis and dot/slot blot hybridization (see, for example,Ausubel (1995) at pages 4-1 to 4-27, and Wu et al. (eds.), “Analysis ofGene Expression at the RNA Level,” in Methods in Gene Biotechnology,pages 225-239 (CRC Press, Inc. 1997)). Nucleic acid probes can bedetectably labeled with radioisotopes such as ³²P or ³⁵S. Alternatively,Zcytor 18 RNA can be detected with a nonradioactive hybridization method(see, for example, Isaac (ed.), Protocols for Nucleic Acid Analysis byNonradioactive Probes (Humana Press, Inc. 1993)). Typically,nonradioactive detection is achieved by enzymatic conversion ofchromogenic or chemiluminescent substrates. Illustrative nonradioactivemoieties include biotin, fluorescein, and digoxigenin.

[0240] Zcytor 18 oligonucleotide probes are also useful for in vivodiagnosis. As an illustration, ¹⁸F-labeled oligonucleotides can beadministered to a subject and visualized by positron emission tomography(Tavitian et al., Nature Medicine 4:467 (1998)).

[0241] Numerous diagnostic procedures take advantage of the polymerasechain reaction (PCR) to increase sensitivity of detection methods.Standard techniques for performing PCR are well-known (see, generally,Mathew (ed.), Protocols in Human Molecular Genetics (Humana Press, Inc.1991), White (ed.), PCR Protocols: Current Methods and Applications(Humana Press, Inc. 1993), Cotter (ed.), Molecular Diagnosis of Cancer(Humana Press, Inc. 1996), Hanausek and Walaszek (eds.), Tumor MarkerProtocols (Humana Press, Inc. 1998), Lo (ed.), Clinical Applications ofPCR (Humana Press, Inc. 1998), and Meltzer (ed.), PCR in Bioanalysis(Humana Press, Inc. 1998)).

[0242] PCR primers can be designed to amplify a portion of the Zcytor 18gene that has a low sequence similarity to a comparable region in otherproteins, such as other cytokine receptor proteins.

[0243] One variation of PCR for diagnostic assays is reversetranscriptase-PCR (RT-PCR). In the RT-PCR technique, RNA is isolatedfrom a biological sample, reverse transcribed to cDNA, and the cDNA isincubated with Zcytor 18 primers (see, for example, Wu et al. (eds.),“Rapid Isolation of Specific cDNAs or Genes by PCR,” in Methods in GeneBiotechnology, pages 15-28 (CRC Press, Inc. 1997)). PCR is thenperformed and the products are analyzed using standard techniques.

[0244] As an illustration, RNA is isolated from biological sample using,for example, the guanidinium-thiocyanate cell lysis procedure describedabove. Alternatively, a solid-phase technique can be used to isolatemRNA from a cell lysate. A reverse transcription reaction can be primedwith the isolated RNA using random oligonucleotides, short homopolymersof dT, or Zcytor 18 anti-sense oligomers. OligodT primers offer theadvantage that various mRNA nucleotide sequences are amplified that canprovide control target sequences. Zcytor 18 sequences are amplified bythe polymerase chain reaction using two flanking oligonucleotide primersthat are typically 20 bases in length.

[0245] PCR amplification products can be detected using a variety ofapproaches. For example, PCR products can be fractionated by gelelectrophoresis, and visualized by ethidium bromide staining.Alternatively, fractionated PCR products can be transferred to amembrane, hybridized with a detectably-labeled Zcytor 18 probe, andexamined by autoradiography. Additional alternative approaches includethe use of digoxigenin-labeled deoxyribonucleic acid triphosphates toprovide chemiluminescence detection, and the C-TRAK colorimetric assay.

[0246] Another approach for detection of Zcytor 18 expression is cyclingprobe technology, in which a single-stranded DNA target binds with anexcess of DNA-RNADNA chimeric probe to form a complex, the RNA portionis cleaved with RNAase H, and the presence of cleaved chimeric probe isdetected (see, for example, Beggs et al., J. Clin. Microbiol. 34:2985(1996), Bekkaoui et al., Biotechniques 20:240 (1996)). Alternativemethods for detection of Zcytor 18 sequences can utilize approaches suchas nucleic acid sequence-based amplification, cooperative amplificationof templates by cross-hybridization, and the ligase chain reaction (see,for example, Marshall et al., U.S. Pat. No. 5,686,272 (1997), Dyer etal., J. Virol. Methods 60:161 (1996), Ehricht et al., Eur. J. Biochem.243:358 (1997), and Chadwick et al., J. Virol. Methods 70:59 (1998)).Other standard methods are known to those of skill in the art.

[0247] Zcytor 18 probes and primers can also be used to detect and tolocalize Zcytor 18 gene expression in tissue samples. Methods for suchin situ hybridization are well-known to those of skill in the art (see,for example, Choo (ed.), In Situ Hybridization Protocols (Humana Press,Inc. 1994), Wu et al. (eds.), “Analysis of Cellular DNA or Abundance ofmRNA by Radioactive In Situ Hybridization (RISH),” in Methods in GeneBiotechnology, pages 259-278 (CRC Press, Inc. 1997), and Wu et al.(eds.), “Localization of DNA or Abundance of mRNA by Fluorescence InSitu Hybridization (RISH),” in Methods in Gene Biotechnology, pages279-289 (CRC Press, Inc. 1997)). Various additional diagnosticapproaches are well-known to those of skill in the art (see, forexample, Mathew (ed.), Protocols in Human Molecular Genetics (HumanaPress, Inc. 1991), Coleman and Tsongalis, Molecular Diagnostics (HumanaPress, Inc. 1996), and Elles, Molecular Diagnosis of Genetic Diseases(Humana Press, Inc., 1996)). Suitable test samples include blood, urine,saliva, tissue biopsy, and autopsy material.

[0248] The Zcytor 18 gene resides in chromosome 3. Thus, nucleic acidprobes that encode Zcytor 18, or a fragment thereof, can be used todetect gross aberrations in chromosome 3. As an example, monosomy 3,trisomy 3, and other structural aberrations of chromosome 3 areassociated with acute myelocytic leukemia, chronic leukemia,neuroendocrine cancer, polyclonal B-cell lymphocytosis, and renal cellcarcinoma (see, for example, Callet-Bauchu et al., Genes, Chromosomes,Cancer 26:221 (1999); Hughson et al., Mod. Pathol. 12:301 (1999); Russoet al., Br. J. Haematol. 105:989 (1999); Lindquist et al., Leukemia14:112 (2000)). Moreover, the Zcytor 18 gene resides in chromosome3p14.3, a region that is associated with various diseases and disorders,including Wernicke-Korsakoff Syndrome, and Bardet-Biedl Syndrome 3.

[0249] In addition, mutations of cytokine receptors are associated withparticular diseases. For example, polymorphisms of cytokine receptorsare associated with pulmonary alveolar proteinosis, familial periodicfever, and erythroleukemia. Thus, Zcytor 18 nucleotide sequences can beused in linkage-based testing for various diseases, and to determinewhether a subject's chromosomes contain a mutation in the Zcytor 18gene. Detectable chromosomal aberrations at the Zcytor 18 gene locusinclude, but are not limited to, aneuploidy, gene copy number changes,insertions, deletions, restriction site changes and rearrangements. Ofparticular interest are genetic alterations that inactivate a Zcytor 18gene.

[0250] Aberrations associated with the Zcytor 18 locus can be detectedusing nucleic acid molecules of the present invention by employingmolecular genetic techniques, such as restriction fragment lengthpolymorphism analysis, short tandem repeat analysis employing PCRtechniques, amplification-refractory mutation system analysis,single-strand conformation polymorphism detection, RNase cleavagemethods, denaturing gradient gel electrophoresis, fluorescence-assistedmismatch analysis, and other genetic analysis techniques known in theart (see, for example, Mathew (ed.), Protocols in Human MolecularGenetics (Humana Press, Inc. 1991), Marian, Chest 108:255 (1995),Coleman and Tsongalis, Molecular Diagnostics (Human Press, Inc. 1996),Elles (ed.) Molecular Diagnosis of Genetic Diseases (Humana Press, Inc.1996), Landegren (ed.), Laboratory Protocols for Mutation Detection(Oxford University Press 1996), Birren et al. (eds.), Genome Analysis,Vol. 2: Detecting Genes (Cold Spring Harbor Laboratory Press 1998),Dracopoli et al. (eds.), Current Protocols in Human Genetics (John Wiley& Sons 1998), and Richards and Ward, “Molecular Diagnostic Testing,” inPrinciples of Molecular Medicine, pages 83-88 (Humana Press, Inc.1998)).

[0251] The protein truncation test is also useful for detecting theinactivation of a gene in which translation-terminating mutationsproduce only portions of the encoded protein (see, for example,Stoppa-Lyonnet et al., Blood 91:3920 (1998)). According to thisapproach, RNA is isolated from a biological sample, and used tosynthesize cDNA. PCR is then used to amplify the Zcytor 18 targetsequence and to introduce an RNA polymerase promoter, a translationinitiation sequence, and an in-frame ATG triplet. PCR products aretranscribed using an RNA polymerase, and the transcripts are translatedin vitro with a T7-coupled reticulocyte lysate system. The translationproducts are then fractionated by SDS-PAGE to determine the lengths ofthe translation products. The protein truncation test is described, forexample, by Dracopoli et al. (eds.), Current Protocols in HumanGenetics, pages 9.11.1-9.11.18 (John Wiley & Sons 1998).

[0252] The present invention also contemplates kits for performing adiagnostic assay for Zcytorl18 gene expression or to detect mutations inthe Zcytor 18 gene. Such kits comprise nucleic acid probes, such asdouble-stranded nucleic acid molecules comprising the nucleotidesequence of nucleotides 192 to 652 of SEQ ID NO:1, the nucleotidesequence of SEQ ID NO:1, or a portion thereof, as well assingle-stranded nucleic acid molecules having the complement of thenucleotide sequence of SEQ ID NO:1, or a portion thereof. Probemolecules may be DNA, RNA, oligonucleotides, and the like. Kits maycomprise nucleic acid primers for performing PCR.

[0253] Such kits can contain all the necessary elements to perform anucleic acid diagnostic assay described above. A kit will comprise atleast one container comprising a Zcytor 18 probe or primer. The kit mayalso comprise a second container comprising one or more reagents capableof indicating the presence of Zcytor 18 sequences. Examples of suchindicator reagents include detectable labels such as radioactive labels,fluorochromes, chemiluminescent agents, and the like. A kit may alsocomprise a means for conveying to the user that the Zcytor 18 probes andprimers are used to detect Zcytor 18 gene expression. For example,written instructions may state that the enclosed nucleic acid moleculescan be used to detect either a nucleic acid molecule that encodes Zcytor18, or a nucleic acid molecule having a nucleotide sequence that iscomplementary to a Zcytor 18-encoding nucleotide sequence. The writtenmaterial can be applied directly to a container, or the written materialcan be provided in the form of a packaging insert.

[0254] 10. Use of Anti-Zcytor 18 Antibodies to Detect Zcytor 18

[0255] The present invention contemplates the use of anti-Zcytor 18antibodies to screen biological samples in vitro for the presence ofZcytor 18. In one type of in vitro assay, anti-Zcytor 18 antibodies areused in liquid phase. For example, the presence of Zcytor 18 in abiological sample can be tested by mixing the biological sample with atrace amount of labeled Zcytor 18 and an anti-Zcytor 18 antibody underconditions that promote binding between Zcytor 18 and its antibody.Complexes of Zcytor 18 and anti-Zcytor 18 in the sample can be separatedfrom the reaction mixture by contacting the complex with an immobilizedprotein which binds with the antibody, such as an Fc antibody orStaphylococcus protein A. The concentration of Zcytor 18 in thebiological sample will be inversely proportional to the amount oflabeled Zcytor 18 bound to the antibody and directly related to theamount of free-labeled Zcytor 18. Illustrative biological samplesinclude blood, urine, saliva, tissue biopsy, and autopsy material.

[0256] Alternatively, in vitro assays can be performed in whichanti-Zcytor 18 antibody is bound to a solid-phase carrier. For example,antibody can be attached to a polymer, such as aminodextran, in order tolink the antibody to an insoluble support such as a polymer-coated bead,a plate or a tube. Other suitable in vitro assays will be readilyapparent to those of skill in the art.

[0257] In another approach, anti-Zcytor 18 antibodies can be used todetect Zcytor 18 in tissue sections prepared from a biopsy specimen.Such immunochemical detection can be used to determine the relativeabundance of Zcytor 18 and to determine the distribution of Zcytor 18 inthe examined tissue. General immunochemistry techniques are wellestablished (see, for example, Ponder, “Cell Marking Techniques andTheir Application,” in Mammalian Development: A Practical Approach, Monk(ed.), pages 1!15-38 (IRL Press 1987), Coligan at pages 5.8.1-5.8.8,Ausubel (1995) at pages 14.6.1 to 14.6.13 (Wiley Interscience 1990), andManson (ed.), Methods In Molecular Biology, Vol. 10: ImmunochemicalProtocols (The Humana Press, Inc. 1992)).

[0258] Immunochemical detection can be performed by contacting abiological sample with an anti-Zcytor 18 antibody, and then contactingthe biological sample with a detectably labeled molecule, which binds tothe antibody. For example, the detectably labeled molecule can comprisean antibody moiety that binds to anti-Zcytor 18 antibody. Alternatively,the anti-Zcytor 18 antibody can be conjugated with avidin/streptavidin(or biotin) and the detectably labeled molecule can comprise biotin (oravidin/streptavidin). Numerous variations of this basic technique arewell-known to those of skill in the art.

[0259] Alternatively, an anti-Zcytor 18 antibody can be conjugated witha detectable label to form an anti-Zcytor 18 immunoconjugate. Suitabledetectable labels include, for example, a radioisotope, a fluorescentlabel, a chemiluminescent label, an enzyme label, a bioluminescent labelor colloidal gold. Methods of making and detecting suchdetectably-labeled immunoconjugates are well-known to those of ordinaryskill in the art, and are described in more detail below.

[0260] The detectable label can be a radioisotope that is detected byautoradiography. Isotopes that are particularly useful for the purposeof the present invention are ³H, ¹²⁵I, ¹³¹I, ³⁵S and ¹⁴C.

[0261] Anti-Zcytor 18 immunoconjugates can also be labeled with afluorescent compound. The presence of a fluorescently-labeled antibodyis determined by exposing the immunoconjugate to light of the properwavelength and detecting the resultant fluorescence. Fluorescentlabeling compounds include fluorescein isothiocyanate, rhodamine,phycoerytherin, phycocyanin, allophycocyanin, o-phthaldehyde andfluorescamine.

[0262] Alternatively, anti-Zcytor 18 immunoconjugates can be detectablylabeled by coupling an antibody component to a chemiluminescentcompound. The presence of the chemiluminescent-tagged immunoconjugate isdetermined by detecting the presence of luminescence that arises duringthe course of a chemical reaction. Examples of chemiluminescent labelingcompounds include luminol, isoluminol, an aromatic acridinium ester, animidazole, an acridinium salt and an oxalate ester.

[0263] Similarly, a bioluminescent compound can be used to labelanti-Zcytor 18 immunoconjugates of the present invention.Bioluminescence is a type of chemiluminescence found in biologicalsystems in which a catalytic protein increases the efficiency of thechemiluminescent reaction. The presence of a bioluminescent protein isdetermined by detecting the presence of luminescence. Bioluminescentcompounds that are useful for labeling include luciferin, luciferase andaequorin.

[0264] Alternatively, anti-Zcytor 18 immunoconjugates can be detectablylabeled by linking an anti-Zcytor 18 antibody component to an enzyme.When the anti-Zcytor 18-enzyme conjugate is incubated in the presence ofthe appropriate substrate, the enzyme moiety reacts with the substrateto produce a chemical moiety, which can be detected, for example, byspectrophotometric, fluorometric or visual means. Examples of enzymesthat can be used to detectably label polyspecific immunoconjugatesinclude β-galactosidase, glucose oxidase, peroxidase and alkalinephosphatase.

[0265] Those of skill in the art will know of other suitable labels,which can be employed in accordance with the present invention. Thebinding of marker moieties to anti-Zcytor 18 antibodies can beaccomplished using standard techniques known to the art. Typicalmethodology in this regard is described by Kennedy et al., Clin. Chim.Acta 70:1 (1976), Schurs et al., Clin. Chim. Acta 81:1 (1977), Shih etal., Intl J. Cancer 46:1101 (1990), Stein et al., Cancer Res. 50:1330(1990), and Coligan, supra.

[0266] Moreover, the convenience and versatility of immunochemicaldetection can be enhanced by using anti-Zcytor 18 antibodies that havebeen conjugated with avidin, streptavidin, and biotin (see, for example,Wilchek et al. (eds.), “Avidin-Biotin Technology,” Methods InEnzymology, Vol. 184 (Academic Press 1990), and Bayer et al.,“Immunochemical Applications of Avidin-Biotin Technology,” in Methods InMolecular Biology, Vol. 10, Manson (ed.), pages 149-162 (The HumanaPress, Inc. 1992).

[0267] Methods for performing immunoassays are well-established. See,for example, Cook and Self, “Monoclonal Antibodies in DiagnosticImmunoassays,” in Monoclonal Antibodies: Production, Engineering, andClinical Application, Ritter and Ladyman (eds.), pages 180-208,(Cambridge University Press, 1995), Perry, “The Role of MonoclonalAntibodies in the Advancement of Immunoassay Technology,” in MonoclonalAntibodies: Principles and Applications, Birch and Lennox (eds.), pages107-120 (Wiley-Liss, Inc. 1995), and Diamandis, Immunoassay (AcademicPress, Inc. 1996).

[0268] The present invention also contemplates kits for performing animmunological diagnostic assay for Zcytor 18 gene expression. Such kitscomprise at least one container comprising an anti-Zcytor 18 antibody,or antibody fragment. A kit may also comprise a second containercomprising one or more reagents capable of indicating the presence ofZcytor 18 antibody or antibody fragments. Examples of such indicatorreagents include detectable labels such as a radioactive label, afluorescent label, a chemiluminescent label, an enzyme label, abioluminescent label, colloidal gold, and the like. A kit may alsocomprise a means for conveying to the user that Zcytor 18 antibodies orantibody fragments are used to detect Zcytor 18 protein. For example,written instructions may state that the enclosed antibody or antibodyfragment can be used to detect Zcytor 18. The written material can beapplied directly to a container, or the written material can be providedin the form of a packaging insert.

[0269] 11. Therapeutic Uses of Polypeptides Having Zcytor 18 Activity

[0270] Amino acid sequences having Zcytor 18 activity can be used tomodulate the immune system by binding Zcytor 18 ligand, and thus,preventing the binding of Zcytor 18 ligand with endogenous Zcytor 18receptor. As an illustration, polypeptides having Zcytor 18 activity canbe used to inhibit cell proliferation associated with, for example,psoriasis or the growth of a tumor (e.g., a melanoma). Zcytor 18antagonists, such as anti-Zcytor 18 antibodies, can also be used tomodulate the immune system by inhibiting the binding of Zcytor 18 ligandwith the endogenous Zcytor 18 receptor.

[0271] Accordingly, the present invention includes the use of proteins,polypeptides, and peptides having Zcytor 18 activity (such as Zcytor 18polypeptides, Zcytor 18 analogs (e.g., anti-Zcytor 18 anti-idiotypeantibodies), and Zcytor 18 fusion proteins) to a subject which lacks anadequate amount of Zcytor 18 polypeptide, or which produces an excess ofZcytor 18 ligand. Zcytor 18 antagonists (e.g., anti-Zcytor 18antibodies) can be also used to treat a subject, which produces anexcess of either Zcytor 18 ligand or Zcytor 18. These molecules can beadministered to any subject in need of treatment, and the presentinvention contemplates both veterinary and human therapeutic uses.Illustrative subjects include mammalian subjects, such as farm animals,domestic animals, and human patients. Human or murine Zcytor 18polypeptides can be used for these applications.

[0272] Generally, the dosage of administered Zcytor 18 (or Zcytor 18analog or fusion protein) will vary depending upon such factors as thesubject's age, weight, height, sex, general medical condition andprevious medical history. Typically, it is desirable to provide therecipient with a dosage of Zcytor 18 polypeptide, which is in the rangeof from about 1 μg/kg to 10 mg/kg (amount of agent/body weight ofsubject), although a lower or higher dosage also may be administered ascircumstances dictate.

[0273] Administration of a Zcytor 18 polypeptide to a subject can beintravenous, intraarterial, intraperitoneal, intramuscular,subcutaneous, intrapleural, intrathecal, by perfusion through a regionalcatheter, or by direct intralesional injection. When administeringtherapeutic proteins by injection, the administration may be bycontinuous infusion or by single or multiple boluses.

[0274] Additional routes of administration include oral,mucosal-membrane, pulmonary, and transcutaneous. Oral delivery issuitable for polyester microspheres, zein microspheres, proteinoidmicrospheres, polycyanoacrylate microspheres, and lipidbased systems(see, for example, DiBase and Morrel, “Oral Delivery ofMicroencapsulated Proteins,” in Protein Delivery: Physical Systems,Sanders and Hendren (eds.), pages 255-288 (Plenum Press 1997)). Thefeasibility of an intranasal delivery is exemplified by such a mode ofinsulin administration (see, for example, Hinchcliffe and Illum, Adv.Drug Deliv. Rev. 35:199 (1999)). Dry or liquid particles comprisingZcytor 18 can be prepared and inhaled with the aid of dry-powderdispersers, liquid aerosol generators, or nebulizers (e.g., Pettit andGombotz, TIBTECH 16:343 (1998); Patton et al., Adv. Drug Deliv. Rev.35:235 (1999)). This approach is illustrated by the AERX diabetesmanagement system, which is a hand-held electronic inhaler that deliversaerosolized insulin into the lungs. Studies have shown that proteins aslarge as 48,000 kDa have been delivered across skin at therapeuticconcentrations with the aid of low-frequency ultrasound, whichillustrates the feasibility of trascutaneous administration (Mitragotriet al., Science 269:850 (1995)). Transdermal delivery usingelectroporation provides another means to administer a molecule havingZcytor 18 binding activity (Potts et al., Pharm. Biotechnol. 10:213(1997)).

[0275] A pharmaceutical composition comprising a protein, polypeptide,or peptide having Zcytor 18 binding activity can be formulated accordingto known methods to prepare pharmaceutically useful compositions,whereby the therapeutic proteins are combined in a mixture with apharmaceutically acceptable carrier. A composition is said to be a“pharmaceutically acceptable carrier” if its administration can betolerated by a recipient patient. Sterile phosphate-buffered saline isone example of a pharmaceutically acceptable carrier. Other suitablecarriers are well-known to those in the art. See, for example, Gennaro(ed.), Remington's Pharmaceutical Sciences, 19th Edition (MackPublishing Company 1995).

[0276] For purposes of therapy, molecules having Zcytor 18 bindingactivity and a pharmaceutically acceptable carrier are administered to apatient in a therapeutically effective amount. A combination of aprotein, polypeptide, or peptide having Zcytor 18 binding activity and apharmaceutically acceptable carrier is said to be administered in a“therapeutically effective amount” if the amount administered isphysiologically significant. An agent is physiologically significant ifits presence results in a detectable change in the physiology of arecipient patient. For example, an agent used to treat inflammation isphysiologically significant if its presence alleviates the inflammatoryresponse. As another example, an agent used to inhibit the growth oftumor cells is physiologically significant if the administration of theagent results in a decrease in the number of tumor cells, decreasedmetastasis, a decrease in the size of a solid tumor, or increasednecrosis of a tumor.

[0277] A pharmaceutical composition comprising Zcytor 18 (or Zcytor 18analog or fusion protein) can be furnished in liquid form, in anaerosol, or in solid form. Liquid forms, are illustrated by injectablesolutions and oral suspensions. Exemplary solid forms include capsules,tablets, and controlled-release forms. The latter form is illustrated byminiosmotic pumps and implants (Bremer et al., Pharm. Biotechnol. 10:239(1997); Ranade, “Implants in Drug Delivery,” in Drug Delivery Systems,Ranade and Hollinger (eds.), pages 95-123 (CRC Press 1995); Bremer etal., “Protein Delivery with Infusion Pumps,” in Protein Delivery:Physical Systems, Sanders and Hendren (eds.), pages 239-254 (PlenumPress 1997); Yewey et al., “Delivery of Proteins from a ControlledRelease Injectable Implant,” in Protein Delivery: Physical Systems,Sanders and Hendren (eds.), pages 93-117 (Plenum Press 1997)).

[0278] Liposomes provide one means to deliver therapeutic polypeptidesto a subject intravenously, intraperitoneally, intrathecally,intramuscularly, subcutaneously, or via oral administration, inhalation,or intranasal administration. Liposomes are microscopic vesicles thatconsist of one or more lipid bilayers surrounding aqueous compartments(see, generally, Bakker-Woudenberg et al., Eur. J. Clin. Microbiol.Infect. Dis. 12 (Suppl. 1):S61 (1993), Kim, Drugs 46:618 (1993), andRanade, “Site-Specific Drug Delivery Using Liposomes as Carriers,” inDrug Delivery Systems, Ranade and Hollinger (eds.), pages 3-24 (CRCPress 1995)). Liposomes are similar in composition to cellular membranesand as a result, liposomes can be administered safely and arebiodegradable. Depending on the method of preparation, liposomes may beunilamellar or multilamellar, and liposomes can vary in size withdiameters ranging from 0.02 μm to greater than 10 μm. A variety ofagents can be encapsulated in liposomes: hydrophobic agents partition inthe bilayers and hydrophilic agents partition within the inner aqueousspace(s) (see, for example, Machy et al., Liposomes In Cell Biology AndPharmacology (John Libbey 1987), and Ostro et al., American J. Hosp.Pharm. 46:1576 (1989)). Moreover, it is possible to control thetherapeutic availability of the encapsulated agent by varying liposomesize, the number of bilayers, lipid composition, as well as the chargeand surface characteristics of the liposomes.

[0279] Liposomes can adsorb to virtually any type of cell and thenslowly release the encapsulated agent. Alternatively, an absorbedliposome may be endocytosed by cells that are phagocytic. Endocytosis isfollowed by intralysosomal degradation of liposomal lipids and releaseof the encapsulated agents (Scherphof et al., Ann. N.Y. Acad. Sci.446:368 (1985)). After intravenous administration, small liposomes (0.1to 1.0 μm) are typically taken up by cells of the reticuloendothelialsystem, located principally in the liver and spleen, whereas liposomeslarger than 3.0 μm are deposited in the lung. This preferential uptakeof smaller liposomes by the cells of the reticuloendothelial system hasbeen used to deliver chemotherapeutic agents to macrophages and totumors of the liver.

[0280] The reticuloendothelial system can be circumvented by severalmethods including saturation with large doses of liposome particles, orselective macrophage inactivation by pharmacological means (Claassen etal., Biochim. Biophys. Acta 802:428 (1984)). In addition, incorporationof glycolipid- or polyethelene glycolderivatized phospholipids intoliposome membranes has been shown to result in a significantly reduceduptake by the reticuloendothelial system (Allen et al., Biochim.Biophys. Acta 1068:133 (1991); Allen et al., Biochim. Biophys. Acta1150:9 (1993)).

[0281] Liposomes can also be prepared to target particular cells ororgans by varying phospholipid composition or by inserting receptors orligands into the liposomes. For example, liposomes, prepared with a highcontent of a nonionic surfactant, have been used to target the liver(Hayakawa et al., Japanese Patent 04-244,018; Kato et al., Biol. Pharm.Bull. 16:960 (1993)). These formulations were prepared by mixing soybeanphospatidylcholine, α-tocopherol, and ethoxylated hydrogenated castoroil (HCO-60) in methanol, concentrating the mixture under vacuum, andthen reconstituting the mixture with water. A liposomal formulation ofdipalmitoylphosphatidylcholine (DPPC) with a soybean-derivedsterylglucoside mixture (SG) and cholesterol (Ch) has also been shown totarget the liver (Shimizu et al., Biol. Pharm. Bull. 20:881 (1997)).

[0282] Alternatively, various targeting ligands can be bound to thesurface of the liposome, such as antibodies, antibody fragments,carbohydrates, vitamins, and transport proteins. For example, liposomescan be modified with branched type galactosyllipid derivatives to targetasialoglycoprotein (galactose) receptors, which are exclusivelyexpressed on the surface of liver cells (Kato and Sugiyama, Crit. Rev.Ther. Drug Carrier Syst. 14:287 (1997); Murahashi et al., Biol. Pharm.Bull. 20:259 (1997)). Similarly, Wu et al., Hepatology 27:772 (1998),have shown that labeling liposomes with asialofetuin led to a shortenedliposome plasma half-life and greatly enhanced uptake ofasialofetuin-labeled liposome by hepatocytes. On the other hand, hepaticaccumulation of liposomes comprising branched type galactosyllipidderivatives can be inhibited by preinjection of asialofetuin (Murahashiet al., Biol. Pharm. Bull.20:259 (1997)). Polyaconitylated human serumalbumin liposomes provide another approach for targeting liposomes toliver cells (Kamps et al., Proc. Nat'l Acad. Sci. USA 94:11681 (1997)).Moreover, Geho, et al. U.S. Pat. No. 4,603,044, describe ahepatocyte-directed liposome vesicle delivery system, which hasspecificity for hepatobiliary receptors associated with the specializedmetabolic cells of the liver.

[0283] In a more general approach to tissue targeting, target cells areprelabeled with biotinylated antibodies specific for a ligand expressedby the target cell (Harasym et al., Adv. Drug Deliv. Rev. 32:99 (1998)).After plasma elimination of free antibody, streptavidin-conjugatedliposomes are administered. In another approach, targeting antibodiesare directly attached to liposomes (Harasym et al., Adv. Drug Deliv.Rev. 32:99 (1998)).

[0284] Polypeptides having Zcytor 18 binding activity can beencapsulated within liposomes using standard techniques of proteinmicroencapsulation (see, for example, Anderson et al., Infect. Immun.31:1099 (1981), Anderson et al., Cancer Res. 50:1853 (1990), and Cohenet al., Biochim. Biophys. Acta 1063:95 (1991), Alving et al.“Preparation and Use of Liposomes in Immunological Studies,” in LiposomeTechnology, 2nd Edition, Vol. III, Gregoriadis (ed.), page 317 (CRCPress 1993), Wassef et al., Meth. Enzymol. 149:124 (1987)). As notedabove, therapeutically useful liposomes may contain a variety ofcomponents. For example, liposomes may comprise lipid derivatives ofpoly(ethylene glycol) (Allen et al., Biochim. Biophys. Acta 1150:9(1993)).

[0285] Degradable polymer microspheres have been designed to maintainhigh systemic levels of therapeutic proteins. Microspheres are preparedfrom degradable polymers such as poly(lactide-co-glycolide) (PLG),polyanhydrides, poly (ortho esters), nonbiodegradable ethylvinyl acetatepolymers, in which proteins are entrapped in the polymer (Gombotz andPettit, Bioconjugate Chem. 6:332 (1995); Ranade, “Role of Polymers inDrug Delivery,” in Drug Delivery Systems, Ranade and Hollinger (eds.),pages 51-93 (CRC Press 1995); Roskos and Maskiewicz, “DegradableControlled Release Systems Useful for Protein Delivery,” in ProteinDelivery: Physical Systems, Sanders and Hendren (eds.), pages 45-92(Plenum Press 1997); Bartus et al., Science 281:1161 (1998); Putney andBurke, Nature Biotechnology 16:153 (1998); Putney, Curr. Opin. Chem.Biol. 2:548 (1998)). Polyethylene glycol (PEG)-coated nanospheres canalso provide carriers for intravenous administration of therapeuticproteins (see, for example, Gref et al., Pharm. Biotechnol. 10:167(1997)).

[0286] The present invention also contemplates chemically modifiedpolypeptides having binding Zcytor 18 activity and Zcytor 18antagonists, in which a polypeptide is linked with a polymer, asdiscussed above.

[0287] Other dosage forms can be devised by those skilled in the art, asshown, for example, by Ansel and Popovich, Pharmaceutical Dosage Formsand Drug Delivery Systems, 5^(th) Edition (Lea & Febiger 1990), Gennaro(ed.), Remington's Pharmaceutical Sciences, 19^(th) Edition (MackPublishing Company 1995), and by Ranade and Hollinger, Drug DeliverySystems (CRC Press 1996).

[0288] As an illustration, pharmaceutical compositions may be suppliedas a kit comprising a container that comprises a polypeptide with aZcytor 18 extracellular domain or a Zcytor 18 antagonist (e.g., anantibody or antibody fragment that binds a Zcytor 18 polypeptide).Therapeutic polypeptides can be provided in the form of an injectablesolution for single or multiple doses, or as a sterile powder that willbe reconstituted before injection. Alternatively, such a kit can includea dry-powder disperser, liquid aerosol generator, or nebulizer foradministration of a therapeutic polypeptide. Such a kit may furthercomprise written information on indications and usage of thepharmaceutical composition. Moreover, such information may include astatement that the Zcytor 18 composition is contraindicated in patientswith known hypersensitivity to Zcytor 18.

[0289] 12. Therapeutic Us s of Zcytor 18 Nucleotide Sequences

[0290] The present invention includes the use of Zcytor 18 nucleotidesequences to provide Zcytor 18 to a subject in need of such treatment.In addition, a therapeutic expression vector can be provided thatinhibits Zcytor 18 gene expression, such as an anti-sense molecule, aribozyme, or an external guide sequence molecule.

[0291] There are numerous approaches to introduce a Zcytor 18 gene to asubject, including the use of recombinant host cells that express Zcytor18, delivery of naked nucleic acid encoding Zcytor 18, use of a cationiclipid carrier with a nucleic acid molecule that encodes Zcytor 18, andthe use of viruses that express Zcytor 18, such as recombinantretroviruses, recombinant adeno-associated viruses, recombinantadenoviruses, and recombinant Herpes simplex viruses (see, for example,Mulligan, Science 260:926 (1993), Rosenberg et al., Science 242:1575(1988), LaSalle et al., Science 259:988 (1993), Wolff et al., Science247:1465 (1990), Breakfield and Deluca, The New Biologist 3:203 (1991)).In an ex vivo approach, for example, cells are isolated from a subject,transfected with a vector that expresses a Zcytor 18 gene, and thentransplanted into the subject.

[0292] In order to effect expression of a Zcytor 18 gene, an expressionvector is constructed in which a nucleotide sequence encoding a Zcytor18 gene is operably linked to a core promoter, and optionally aregulatory element, to control gene transcription. The generalrequirements of an expression vector are described above.

[0293] Alternatively, a Zcytor 18 gene can be delivered usingrecombinant viral vectors, including for example, adenoviral vectors(e.g., Kass-Eisler et al., Proc. Nat'l Acad. Sci. USA 90:11498 (1993),Kolls et al., Proc. Nat'l Acad. Sci. USA 91:215 (1994), Li et al., Hum.Gene Ther. 4:403 (1993), Vincent et al., Nat. Genet. 5:130 (1993), andZabner et al., Cell 75:207 (1993)), adenovirus-associated viral vectors(Flotte et al., Proc. Nat'l Acad. Sci. USA 90:10613 (1993)),alphaviruses such as Semliki Forest Virus and Sindbis Virus (Hertz andHuang, J. Vir. 66:857 (1992), Raju and Huang, J. Vir. 65:2501 (1991),and xiong et al., Science 243:1188 (1989)), herpes viral vectors (e.g.,U.S. Pat. Nos. 4,769,331, 4,859,587, 5,288,641 and 5,328,688),parvovirus vectors (Koering et al., Hum. Gene Therap. 5:457 (1994)), poxvirus vectors (Ozaki et al., Biochem. Biophys. Res. Comm. 193:653(1993), Panicali and Paoletti, Proc. Nat'l Acad. Sci. USA 79:4927(1982)), pox viruses, such as canary pox virus or vaccinia virus(Fisher-Hoch et al., Proc. Nat'l Acad. Sci. USA 86:317 (1989), andFlexner et al., Ann. N.Y. Acad. Sci. 569:86 (1989)), and retroviruses(e.g., Baba et al., J. Neurosurg 79:729 (1993), Ram et al., Cancer Res.53:83 (1993), Takamiya et al., J. Neurosci. Res 33:493 (1992), Vile andHart, Cancer Res. 53:962 (1993), Vile and Hart, Cancer Res. 53:3860(1993), and Anderson et al., U.S. Pat. No. 5,399,346). Within variousembodiments, either the viral vector itself, or a viral particle whichcontains the viral vector may be utilized in the methods andcompositions described below.

[0294] As an illustration of one system, adenovirus, a double-strandedDNA virus, is a well-characterized gene transfer vector for delivery ofa heterologous nucleic acid molecule (for a review, see Becker et al.,Meth. Cell Biol. 43:161 (1994); Douglas and Curiel, Science & Medicine4:44 (1997)). The adenovirus system offers several advantages including:(i) the ability to accommodate relatively large DNA inserts, (ii) theability to be grown to high-titer, (iii) the ability to infect a broadrange of mammalian cell types, and (iv) the ability to be used with manydifferent promoters including ubiquitous, tissue specific, andregulatable promoters. In addition, adenoviruses can be administered byintravenous injection, because the viruses are stable in thebloodstream.

[0295] Using adenovirus vectors where portions of the adenovirus genomeare deleted, inserts are incorporated into the viral DNA by directligation or by homologous recombination with a co-transfected plasmid.In an exemplary system, the essential E1 gene is deleted from the viralvector, and the virus will not replicate unless the E1 gene is providedby the host cell. When intravenously administered to intact animals,adenovirus primarily targets the liver. Although an adenoviral deliverysystem with an E1 gene deletion cannot replicate in the host cells, thehost's tissue will express and process an encoded heterologous protein.Host cells will also secrete the heterologous protein if thecorresponding gene includes a secretory signal sequence. Secretedproteins will enter the circulation from tissue that expresses theheterologous gene (e.g., the highly vascularized liver).

[0296] Moreover, adenoviral vectors containing various deletions ofviral genes can be used to reduce or eliminate immune responses to thevector. Such adenoviruses are E1-deleted, and in addition, containdeletions of E2A or E4 (Lusky et al., J. Virol. 72:2022 (1998); Raper etal., Human Gene Therapy 9:671 (1998)). The deletion of E2b has also beenreported to reduce immune responses (Amalfitano et al., J. Virol. 72:926(1998)). By deleting the entire adenovirus genome, very large inserts ofheterologous DNA can be accommodated. The generation of so called“gutless” adenoviruses, where all viral genes are deleted, isparticularly advantageous for insertion of large inserts of heterologousDNA (for a review, see Yeh. and Perricaudet, FASEB J. 11:615 (1997)).

[0297] High titer stocks of recombinant viruses capable of expressing atherapeutic gene can be obtained from infected mammalian cells usingstandard methods. For example, recombinant herpes simplex virus can beprepared in Vero cells, as described by Brandt et al., J. Gen. Virol.72:2043 (1991), Herold et al., J. Gen. Virol. 75:1211 (1994), Visalliand Brandt, Virology 185:419 (1991), Grau et al., Invest. Ophthalmol.Vis. Sci. 30:2474 (1989), Brandt et al., J. Virol. Meth. 36:209 (1992),and by Brown and MacLean (eds.), HSV Virus Protocols (Humana Press1997).

[0298] Alternatively, an expression vector comprising a Zcytor 18 genecan be introduced into a subject's cells by lipofection in vivo usingliposomes. Synthetic cationic lipids can be used to prepare liposomesfor in vivo transfection of a gene encoding a marker (Feigner et al.,Proc. Nat'l Acad. Sci. USA 84:7413 (1987); Mackey et al., Proc. Nat'lAcad. Sci. USA 85:8027 (1988)). The use of lipofection to introduceexogenous genes into specific organs in vivo has certain practicaladvantages. Liposomes can be used to direct transfection to particularcell types, which is particularly advantageous in a tissue with cellularheterogeneity, such as the pancreas, liver, kidney, and brain. Lipidsmay be chemically coupled to other molecules for the purpose oftargeting. Targeted peptides (e.g., hormones or neurotransmitters),proteins such as antibodies, or non-peptide molecules can be coupled toliposomes chemically.

[0299] Electroporation is another alternative mode of administration.For example, Aihara and Miyazaki, Nature Biotechnology 16:867 (1998),have demonstrated the use of in vivo electroporation for gene transferinto muscle.

[0300] In an alternative approach to gene therapy, a therapeutic genemay encode a Zcytor 18 anti-sense RNA that inhibits the expression ofZcytor 18. Suitable sequences for anti-sense molecules can be derivedfrom the nucleotide sequences of Zcytor 18 disclosed herein.

[0301] Alternatively, an expression vector can be constructed in which aregulatory element is operably linked to a nucleotide sequence thatencodes a ribozyme. Ribozymes can be designed to express endonucleaseactivity that is directed to a certain target sequence in an mRNAmolecule (see, for example, Draper and Macejak, U.S. Pat. No. 5,496,698,McSwiggen, U.S. Pat. No. 5,525,468, Chowrira and McSwiggen, U.S. Pat.No. 5,631,359, and Robertson and Goldberg, U.S. Pat. No. 5,225,337). Inthe context of the present invention, ribozymes include nucleotidesequences that bind with Zcytor 18 mRNA.

[0302] In another approach, expression vectors can be constructed inwhich a regulatory element directs the production of RNA transcriptscapable of promoting RNase P-mediated cleavage of mRNA molecules thatencode a Zcytor 18 gene. According to this approach, an external guidesequence can be constructed for directing the endogenous ribozyme, RNaseP, to a particular species of intracellular mRNA, which is subsequentlycleaved by the cellular ribozyme (see, for example, Altman et al., U.S.Pat. No. 5,168,053, Yuan et al., Science 263:1269 (1994), Pace et al.,international publication No. WO 96/18733, George et al., internationalpublication No. WO 96/21731, and Werner et al., internationalpublication No. WO 97/33991). For example, the external guide sequencecan comprise a ten to fifteen nucleotide sequence complementary toZcytor 18 mRNA, and a 3′-NCCA nucleotide sequence, wherein N ispreferably a purine. The external guide sequence transcripts bind to thetargeted mRNA species by the formation of base pairs between the mRNAand the complementary external guide sequences, thus promoting cleavageof mRNA by RNase P at the nucleotide located at the 5′-side of thebase-paired region.

[0303] In general, the dosage of a composition comprising a therapeuticvector having a Zcytor 18 nucleotide sequence, such as a recombinantvirus, will vary depending upon such factors as the subject's age,weight, height, sex, general medical condition and previous medicalhistory. Suitable routes of administration of therapeutic vectorsinclude intravenous injection, intraarterial injection, intraperitonealinjection, intramuscular injection, intratumoral injection, andinjection into a cavity that contains a tumor. As an illustration,Horton et al., Proc. Nat'l Acad. Sci. USA 96:1553 (1999), demonstratedthat intramuscular injection of plasmid DNA encoding interferon-αproduces potent antitumor effects on primary and metastatic tumors in amurine model.

[0304] A composition comprising viral vectors, non-viral vectors, or acombination of viral and non-viral vectors of the present invention canbe formulated according to known methods to prepare pharmaceuticallyuseful compositions, whereby vectors or viruses are combined in amixture with a pharmaceutically acceptable carrier. As noted above, acomposition, such as phosphate-buffered saline is said to be a“pharmaceutically acceptable carrier” if its administration can betolerated by a recipient subject. Other suitable carriers are well-knownto those in the art (see, for example, Remington's PharmaceuticalSciences, 19th Ed. (Mack Publishing Co. 1995), and Gilman's thePharmacological Basis of Therapeutics, 7th Ed. (MacMillan Publishing Co.1985)).

[0305] For purposes of therapy, a therapeutic gene expression vector, ora recombinant virus comprising such a vector, and a pharmaceuticallyacceptable carrier are administered to a subject in a therapeuticallyeffective amount. A combination of an expression vector (or virus) and apharmaceutically acceptable carrier is said to be administered in a“therapeutically effective amount” if the amount administered isphysiologically significant. An agent is physiologically significant ifits presence results in a detectable change in the physiology of arecipient subject. For example, an agent used to treat inflammation isphysiologically significant if its presence alleviates the inflammatoryresponse.

[0306] When the subject treated with a therapeutic gene expressionvector or a recombinant virus is a human, then the therapy is preferablysomatic cell gene therapy. That is, the preferred treatment of a humanwith a therapeutic gene expression vector or a recombinant virus doesnot entail introducing into cells a nucleic acid molecule that can formpart of a human germ line and be passed onto successive generations(i.e., human germ line gene therapy).

[0307] 13. Production of Transgenic Mice

[0308] Transgenic mice can be engineered to over-express the Zcytor 18gene in all tissues or under the control of a tissue-specific ortissue-preferred regulatory element. These over-producers of Zcytor 18can be used to characterize the phenotype that results fromover-expression, and the transgenic animals can serve as models forhuman disease caused by excess Zcytor 18. Transgenic mice thatover-express Zcytor 18 also provide model bioreactors for production ofZcytor 18, such as soluble Zcytor 18, in the milk or blood of largeranimals. Methods for producing transgenic mice are well-known to thoseof skill in the art (see, for example, Jacob, “Expression and Knockoutof Interferons in Transgenic Mice,” in Overexpression and Knockout ofCytokines in Transgenic Mice, Jacob (ed.), pages 111-124 (AcademicPress, Ltd. 1994), Monastersky and Robl (eds.), Strategies in TransgenicAnimal Science (ASM Press 1995), and Abbud and Nilson, “RecombinantProtein Expression in Transgenic Mice,” in Gene Expression Systems:Using Nature for the Art of Expression, Fernandez and Hoeffler (eds.),pages 367-397 (Academic Press, Inc. 1999)).

[0309] For example, a method for producing a transgenic mouse thatexpresses a Zcytor 18 gene can begin with adult, fertile males (studs)(B6C3f1, 2-8 months of age (Taconic Farms, Germantown, N.Y.)),vasectomized males (duds) (B6D2f1, 2-8 months, (Taconic Farms)),prepubescent fertile females (donors) (B6C3f1, 4-5 weeks, (TaconicFarms)) and adult fertile females (recipients) (B6D2f1, 2-4 months,(Taconic Farms)). The donors are acclimated for one week and theninjected with approximately 8 IU/mouse of Pregnant Mare's Serumgonadotrophin (Sigma Chemical Company; St. Louis, Mo.) I.P., and 46-47hours later, 8 IU/mouse of human Chorionic Gonadotropin (hCG (Sigma))I.P. to induce superovulation. Donors are mated with studs subsequent tohormone injections. Ovulation generally occurs within 13 hours of hCGinjection. Copulation is confirmed by the presence of a vaginal plug themorning following mating.

[0310] Fertilized eggs are collected under a surgical scope. Theoviducts are collected and eggs are released into urinanalysis slidescontaining hyaluronidase (Sigma). Eggs are washed once in hyaluronidase,and twice in Whitten's W640 medium (described, for example, by Meninoand O'Claray, Biol. Reprod. 77:159 (1986), and Dienhart and Downs,Zygote 4:129 (1996)) that has been incubated with 5% CO₂, 5% ° and 90% Nat 37° C. The eggs are then stored in a 37° C./5% CO incubator untilmicroinjection.

[0311] Ten to twenty micrograms of plasmid DNA containing a Zcytor 18encoding sequence is linearized, gel-purified, and resuspended in 10 mMTris-HCl (pH 7.4), 0.25 mM EDTA (pH 8.0), at a final concentration of5-10 nanograms per microliter for microinjection. For example, theZcytor 18 encoding sequences can encode a polypeptide comprising aminoacid residues 36 to 313 of SEQ ID NO:2, amino acid residues 36 to 299 ofSEQ ID NO:8, or amino acid residues 36 to 300 of SEQ ID NO:12.

[0312] Plasmid DNA is microinjected into harvested eggs contained in adrop of W640 medium overlaid by warm, CO₂-equilibrated mineral oil. TheDNA is drawn into an injection needle (pulled from a 0.75 mm ID, 1 mm ODborosilicate glass capillary), and injected into individual eggs. Eachegg is penetrated with the injection needle, into one or both of thehaploid pronuclei.

[0313] Picoliters of DNA are injected into the pronuclei, and theinjection needle withdrawn without coming into contact with thenucleoli. The procedure is repeated until all the eggs are injected.Successfully microinjected eggs are transferred into an organtissue-culture dish with pre-gassed W640 medium for storage overnight ina 37° C./5% CO₂ incubator.

[0314] The following day, two-cell embryos are transferred intopseudopregnant recipients. The recipients are identified by the presenceof copulation plugs, after copulating with vasectomized duds. Recipientsare anesthetized and shaved on the dorsal left side and transferred to asurgical microscope. A small incision is made in the skin and throughthe muscle wall in the middle of the abdominal area outlined by theribcage, the saddle, and the hind leg, midway between knee and spleen.The reproductive organs are exteriorized onto a small surgical drape.The fat pad is stretched out over the surgical drape, and a babyserrefine (Roboz, Rockville, Md.) is attached to the fat pad and lefthanging over the back of the mouse, preventing the organs from slidingback in.

[0315] With a fine transfer pipette containing mineral oil followed byalternating W640 and air bubbles, 12-17 healthy two-cell embryos fromthe previous day's injection are transferred into the recipient. Theswollen ampulla is located and holding the oviduct between the ampullaand the bursa, a nick in the oviduct is made with a 28 g needle close tothe bursa, making sure not to tear the ampulla or the bursa.

[0316] The pipette is transferred into the nick in the oviduct, and theembryos are blown in, allowing the first air bubble to escape thepipette. The fat pad is.gently pushed into the peritoneum, and thereproductive organs allowed to slide in. The peritoneal wall is closedwith one suture and the skin closed with a wound clip. The micerecuperate on a 37° C. slide warmer for a minimum of four hours.

[0317] The recipients are returned to cages in pairs, and allowed 19-21days gestation. After birth, 19-21 days postpartum is allowed beforeweaning. The weanlings are sexed and placed into separate sex cages, anda 0.5 cm biopsy (used for genotyping) is snipped off the tail with cleanscissors.

[0318] Genomic DNA is prepared from the tail snips using, for example, aQIAGEN DNEASY kit following the manufacturer's instructions. Genomic DNAis analyzed by PCR using primers designed to amplify a Zcytor 18 gene ora selectable marker gene that was introduced in the same plasmid. Afteranimals are confirmed to be transgenic, they are back-crossed into aninbred strain by placing a transgenic female with a wild-type male, or atransgenic male with one or two wild-type female(s). As pups are bornand weaned, the sexes are separated, and their tails snipped forgenotyping.

[0319] To check for expression of a transgene in a live animal, apartial hepatectomy is performed. A surgical prep is made of the upperabdomen directly below the zyphoid process. Using sterile technique, asmall 1.5-2 cm incision is made below the sternum and the left laterallobe of the liver exteriorized. Using 4-0 silk, a tie is made around thelower lobe securing it outside the body cavity. An atraumatic clamp isused to hold the tie while a second loop of absorbable Dexon (AmericanCyanamid; Wayne, N.J.) is placed proximal to the first tie. A distal cutis made from the Dexon tie and approximately 100 mg of the excised livertissue is placed in a sterile petri dish. The excised liver section istransferred to a 14 ml polypropylene round bottom tube and snap frozenin liquid nitrogen and then stored on dry ice. The surgical site isclosed with suture and wound clips, and the animal's cage placed on a37° C. heating pad for 24 hours post operatively. The animal is checkeddaily post operatively and the wound clips removed 7-10 days aftersurgery. The expression level of Zcytor 18 mRNA is examined for eachtransgenic mouse using an RNA solution hybridization assay or polymerasechain reaction.

[0320] In addition to producing transgenic mice that over-express Zcytor18, it is useful to engineer transgenic mice with either abnormally lowor no expression of the gene. Such transgenic mice provide useful modelsfor diseases associated with a lack of Zcytor 18. As discussed above,Zcytor 18 gene expression can be inhibited using anti-sense genes,ribozyme genes, or external guide sequence genes. To produce transgenicmice that under-express the Zcytor 18 gene, such inhibitory sequencesare targeted to Zcytor 18 mRNA. Methods for producing transgenic micethat have abnormally low expression of a particular gene are known tothose in the art (see, for example, Wu et al., “Gene Underexpression inCultured Cells and Animals by Antisense DNA and RNA Strategies,” inMethods in Gene Biotechnology, pages 205-224 (CRC Press 1997)).

[0321] An alternative approach to producing transgenic mice that havelittle or no Zcytor 18 gene expression is to generate mice having atleast one normal Zcytor 18 allele replaced by a nonfunctional Zcytor 18gene. One method of designing a nonfunctional Zcytor 18 gene is toinsert another gene, such as a selectable marker gene, within a nucleicacid molecule that encodes Zcytor 18. Standard methods for producingthese so-called “knockout mice” are known to those skilled in the art(see, for example, Jacob, “Expression and Knockout of Interferons inTransgenic Mice,” in Overexpression and Knockout of Cytokines inTransgenic Mice, Jacob (ed.), pages 111-124 (Academic Press, Ltd. 1994),and Wu et al., “New Strategies for Gene Knockout,” in Methods in GeneBiotechnology, pages 339-365 (CRC Press 1997)).

[0322] From the foregoing, it will be appreciated that, althoughspecific embodiments of the invention have been described herein forpurposes of illustration, various modifications may be made withoutdeviating from the spirit and scope of the invention. Accordingly, theinvention is not limited except as by the appended claims.

1 13 1 2383 DNA Homo sapiens CDS (86)...(2344) 1 ccgccgcggc caccgcccactcggggctgg ccagcggcgg gcggccgggg cgcagagaac 60 ggcctggctg ggcgagcgcacggcc atg gcc ccg tgg ctg cag ctc tgc tcc 112 Met Ala Pro Trp Leu GlnLeu Cys Ser 1 5 gtc ttc ttt acg gtc aac gcc tgc ctc aac ggc tcg cag ctggct gtg 160 Val Phe Phe Thr Val Asn Ala Cys Leu Asn Gly Ser Gln Leu AlaVal 10 15 20 25 gcc gct ggc ggg tcc ggc cgc gcg cgg ggc gcc gac acc tgtggc tgg 208 Ala Ala Gly Gly Ser Gly Arg Ala Arg Gly Ala Asp Thr Cys GlyTrp 30 35 40 agg atg aaa gcg gct gcc cga ccc cgg ctt tgt gtt gct aat gaggga 256 Arg Met Lys Ala Ala Ala Arg Pro Arg Leu Cys Val Ala Asn Glu Gly45 50 55 gtg ggg cca gcc agc aga aac agt ggg ctg tac aac atc acc ttc aaa304 Val Gly Pro Ala Ser Arg Asn Ser Gly Leu Tyr Asn Ile Thr Phe Lys 6065 70 tat gac aat tgt acc acc tac ttg aat cca gtg ggg aag cat gtg att352 Tyr Asp Asn Cys Thr Thr Tyr Leu Asn Pro Val Gly Lys His Val Ile 7580 85 gct gac gcc cag aat atc acc atc agc cag tat gct tgc cat gac caa400 Ala Asp Ala Gln Asn Ile Thr Ile Ser Gln Tyr Ala Cys His Asp Gln 9095 100 105 gtg gca gtc acc att ctt tgg tcc cca ggg gcc ctc ggc atc gaattc 448 Val Ala Val Thr Ile Leu Trp Ser Pro Gly Ala Leu Gly Ile Glu Phe110 115 120 ctg aaa gga ttt cgg gta ata ctg gag gag ctg aag tcg gag ggaaga 496 Leu Lys Gly Phe Arg Val Ile Leu Glu Glu Leu Lys Ser Glu Gly Arg125 130 135 cag tgc caa caa ctg att cta aag gat ccg aag cag ctc aac agtagc 544 Gln Cys Gln Gln Leu Ile Leu Lys Asp Pro Lys Gln Leu Asn Ser Ser140 145 150 ttc aaa aga act gga atg gaa tct caa cct ttc ctg aat atg aaattt 592 Phe Lys Arg Thr Gly Met Glu Ser Gln Pro Phe Leu Asn Met Lys Phe155 160 165 gaa acg gat tat ttc gta aag gtt gtc cct ttt cct tcc att aaaaac 640 Glu Thr Asp Tyr Phe Val Lys Val Val Pro Phe Pro Ser Ile Lys Asn170 175 180 185 gaa agc aat tac cac cct ttc ttc ttt aga acc cga gcc tgtgac ctg 688 Glu Ser Asn Tyr His Pro Phe Phe Phe Arg Thr Arg Ala Cys AspLeu 190 195 200 ttg tta cag ccg gac aat cta gct tgt aaa ccc ttc tgg aagcct cgg 736 Leu Leu Gln Pro Asp Asn Leu Ala Cys Lys Pro Phe Trp Lys ProArg 205 210 215 aac ctg aac atc agc cag cat ggc tcg gac atg cag gtg tccttc gac 784 Asn Leu Asn Ile Ser Gln His Gly Ser Asp Met Gln Val Ser PheAsp 220 225 230 cat gca ccg cac aac ttc ggc ttc cgt ttc ttc tat ctt cactac aag 832 His Ala Pro His Asn Phe Gly Phe Arg Phe Phe Tyr Leu His TyrLys 235 240 245 ctc aag cac gaa gga cct ttc aag cga aag acc tgt aag caggag caa 880 Leu Lys His Glu Gly Pro Phe Lys Arg Lys Thr Cys Lys Gln GluGln 250 255 260 265 act aca gag acg acc agc tgc ctc ctt caa aat gtt tctcca ggg gat 928 Thr Thr Glu Thr Thr Ser Cys Leu Leu Gln Asn Val Ser ProGly Asp 270 275 280 tat ata att gag ctg gtg gat gac act aac aca aca agaaaa gtg atg 976 Tyr Ile Ile Glu Leu Val Asp Asp Thr Asn Thr Thr Arg LysVal Met 285 290 295 cat tat gcc tta aag cca gtg cac tcc ccg tgg gcc gggccc atc aga 1024 His Tyr Ala Leu Lys Pro Val His Ser Pro Trp Ala Gly ProIle Arg 300 305 310 gcc gtg gcc atc aca gtg cca ctg gta gtc ata tcg gcattc gcg acg 1072 Ala Val Ala Ile Thr Val Pro Leu Val Val Ile Ser Ala PheAla Thr 315 320 325 ctc ttc act gtg atg tgc cgc aag aag caa caa gaa aatata tat tca 1120 Leu Phe Thr Val Met Cys Arg Lys Lys Gln Gln Glu Asn IleTyr Ser 330 335 340 345 cat tta gat gaa gag agc tct gag tct tcc aca tacact gca gca ctc 1168 His Leu Asp Glu Glu Ser Ser Glu Ser Ser Thr Tyr ThrAla Ala Leu 350 355 360 cca aga gag agg ctc cgg ccg cgg ccg aag gtc tttctc tgc tat tcc 1216 Pro Arg Glu Arg Leu Arg Pro Arg Pro Lys Val Phe LeuCys Tyr Ser 365 370 375 agt aaa gat ggc cag aat cac atg aat gtc gtc cagtgt ttc gcc tac 1264 Ser Lys Asp Gly Gln Asn His Met Asn Val Val Gln CysPhe Ala Tyr 380 385 390 ttc ctc cag gac ttc tgt ggc tgt gag gtg gct ctggac ctg tgg gaa 1312 Phe Leu Gln Asp Phe Cys Gly Cys Glu Val Ala Leu AspLeu Trp Glu 395 400 405 gac ttc agc ctc tgt aga gaa ggg cag aga gaa tgggtc atc cag aag 1360 Asp Phe Ser Leu Cys Arg Glu Gly Gln Arg Glu Trp ValIle Gln Lys 410 415 420 425 atc cac gag tcc cag ttc atc att gtg gtt tgttcc aaa ggt atg aag 1408 Ile His Glu Ser Gln Phe Ile Ile Val Val Cys SerLys Gly Met Lys 430 435 440 tac ttt gtg gac aag aag aac tac aaa cac aaagga ggt ggc cga ggc 1456 Tyr Phe Val Asp Lys Lys Asn Tyr Lys His Lys GlyGly Gly Arg Gly 445 450 455 tcg ggg aaa gga gag ctc ttc ctg gtg gcg gtgtca gcc att gcc gaa 1504 Ser Gly Lys Gly Glu Leu Phe Leu Val Ala Val SerAla Ile Ala Glu 460 465 470 aag ctc cgc cag gcc aag cag agt tcg tcc gcggcg ctc agc aag ttt 1552 Lys Leu Arg Gln Ala Lys Gln Ser Ser Ser Ala AlaLeu Ser Lys Phe 475 480 485 atc gcc gtc tac ttt gat tat tcc tgc gag ggagac gtc ccc ggt atc 1600 Ile Ala Val Tyr Phe Asp Tyr Ser Cys Glu Gly AspVal Pro Gly Ile 490 495 500 505 cta gac ctg agt acc aag tac aga ctc atggac aat ctt cct cag ctc 1648 Leu Asp Leu Ser Thr Lys Tyr Arg Leu Met AspAsn Leu Pro Gln Leu 510 515 520 tgt tcc cac ttg cac tcc cga gac cac ggcctc cag gag ccg ggg cag 1696 Cys Ser His Leu His Ser Arg Asp His Gly LeuGln Glu Pro Gly Gln 525 530 535 cac acg cga cag ggc agc aga agg aac tacttc cgg agc aag tca ggc 1744 His Thr Arg Gln Gly Ser Arg Arg Asn Tyr PheArg Ser Lys Ser Gly 540 545 550 cgg tcc cta tac gtc gcc att tgc aac atgcac cag ttt att gac gag 1792 Arg Ser Leu Tyr Val Ala Ile Cys Asn Met HisGln Phe Ile Asp Glu 555 560 565 gag ccc gac tgg ttc gaa aag cag ttc gttccc ttc cat cct cct cca 1840 Glu Pro Asp Trp Phe Glu Lys Gln Phe Val ProPhe His Pro Pro Pro 570 575 580 585 ctg cgc tac cgg gag cca gtc ttg gagaaa ttt gat tcg ggc ttg gtt 1888 Leu Arg Tyr Arg Glu Pro Val Leu Glu LysPhe Asp Ser Gly Leu Val 590 595 600 tta aat gat gtc atg tgc aaa cca gggcct gag agt gac ttc tgc cta 1936 Leu Asn Asp Val Met Cys Lys Pro Gly ProGlu Ser Asp Phe Cys Leu 605 610 615 aag gta gag gcg gct gtt ctt ggg gcaacc gga cca gcc gac tcc cag 1984 Lys Val Glu Ala Ala Val Leu Gly Ala ThrGly Pro Ala Asp Ser Gln 620 625 630 cac gag agt cag cat ggg ggc ctg gaccaa gac ggg gag gcc cgg cct 2032 His Glu Ser Gln His Gly Gly Leu Asp GlnAsp Gly Glu Ala Arg Pro 635 640 645 gcc ctt gac ggt agc gcc gcc ctg caaccc ctg ctg cac acg gtg aaa 2080 Ala Leu Asp Gly Ser Ala Ala Leu Gln ProLeu Leu His Thr Val Lys 650 655 660 665 gcc ggc agc ccc tcg gac atg ccgcgg gac tca ggc atc tat gac tcg 2128 Ala Gly Ser Pro Ser Asp Met Pro ArgAsp Ser Gly Ile Tyr Asp Ser 670 675 680 tct gtg ccc tca tcc gag ctg tctctg cca ctg atg gaa gga ctc tcg 2176 Ser Val Pro Ser Ser Glu Leu Ser LeuPro Leu Met Glu Gly Leu Ser 685 690 695 acg gac cag aca gaa acg tct tccctg acg gag agc gtg tcc tcc tct 2224 Thr Asp Gln Thr Glu Thr Ser Ser LeuThr Glu Ser Val Ser Ser Ser 700 705 710 tca ggc ctg ggt gag gag gaa cctcct gcc ctt cct tcc aag ctc ctc 2272 Ser Gly Leu Gly Glu Glu Glu Pro ProAla Leu Pro Ser Lys Leu Leu 715 720 725 tct tct ggg tca tgc aaa gca gatctt ggt tgc cgc agc tac act gat 2320 Ser Ser Gly Ser Cys Lys Ala Asp LeuGly Cys Arg Ser Tyr Thr Asp 730 735 740 745 gaa ctc cac gcg gtc gcc cctttg taacaaaacg aaagagtcta agcattgcca 2374 Glu Leu His Ala Val Ala ProLeu 750 ctttagctg 2383 2 753 PRT Homo sapiens 2 Met Ala Pro Trp Leu GlnLeu Cys Ser Val Phe Phe Thr Val Asn Ala 1 5 10 15 Cys Leu Asn Gly SerGln Leu Ala Val Ala Ala Gly Gly Ser Gly Arg 20 25 30 Ala Arg Gly Ala AspThr Cys Gly Trp Arg Met Lys Ala Ala Ala Arg 35 40 45 Pro Arg Leu Cys ValAla Asn Glu Gly Val Gly Pro Ala Ser Arg Asn 50 55 60 Ser Gly Leu Tyr AsnIle Thr Phe Lys Tyr Asp Asn Cys Thr Thr Tyr 65 70 75 80 Leu Asn Pro ValGly Lys His Val Ile Ala Asp Ala Gln Asn Ile Thr 85 90 95 Ile Ser Gln TyrAla Cys His Asp Gln Val Ala Val Thr Ile Leu Trp 100 105 110 Ser Pro GlyAla Leu Gly Ile Glu Phe Leu Lys Gly Phe Arg Val Ile 115 120 125 Leu GluGlu Leu Lys Ser Glu Gly Arg Gln Cys Gln Gln Leu Ile Leu 130 135 140 LysAsp Pro Lys Gln Leu Asn Ser Ser Phe Lys Arg Thr Gly Met Glu 145 150 155160 Ser Gln Pro Phe Leu Asn Met Lys Phe Glu Thr Asp Tyr Phe Val Lys 165170 175 Val Val Pro Phe Pro Ser Ile Lys Asn Glu Ser Asn Tyr His Pro Phe180 185 190 Phe Phe Arg Thr Arg Ala Cys Asp Leu Leu Leu Gln Pro Asp AsnLeu 195 200 205 Ala Cys Lys Pro Phe Trp Lys Pro Arg Asn Leu Asn Ile SerGln His 210 215 220 Gly Ser Asp Met Gln Val Ser Phe Asp His Ala Pro HisAsn Phe Gly 225 230 235 240 Phe Arg Phe Phe Tyr Leu His Tyr Lys Leu LysHis Glu Gly Pro Phe 245 250 255 Lys Arg Lys Thr Cys Lys Gln Glu Gln ThrThr Glu Thr Thr Ser Cys 260 265 270 Leu Leu Gln Asn Val Ser Pro Gly AspTyr Ile Ile Glu Leu Val Asp 275 280 285 Asp Thr Asn Thr Thr Arg Lys ValMet His Tyr Ala Leu Lys Pro Val 290 295 300 His Ser Pro Trp Ala Gly ProIle Arg Ala Val Ala Ile Thr Val Pro 305 310 315 320 Leu Val Val Ile SerAla Phe Ala Thr Leu Phe Thr Val Met Cys Arg 325 330 335 Lys Lys Gln GlnGlu Asn Ile Tyr Ser His Leu Asp Glu Glu Ser Ser 340 345 350 Glu Ser SerThr Tyr Thr Ala Ala Leu Pro Arg Glu Arg Leu Arg Pro 355 360 365 Arg ProLys Val Phe Leu Cys Tyr Ser Ser Lys Asp Gly Gln Asn His 370 375 380 MetAsn Val Val Gln Cys Phe Ala Tyr Phe Leu Gln Asp Phe Cys Gly 385 390 395400 Cys Glu Val Ala Leu Asp Leu Trp Glu Asp Phe Ser Leu Cys Arg Glu 405410 415 Gly Gln Arg Glu Trp Val Ile Gln Lys Ile His Glu Ser Gln Phe Ile420 425 430 Ile Val Val Cys Ser Lys Gly Met Lys Tyr Phe Val Asp Lys LysAsn 435 440 445 Tyr Lys His Lys Gly Gly Gly Arg Gly Ser Gly Lys Gly GluLeu Phe 450 455 460 Leu Val Ala Val Ser Ala Ile Ala Glu Lys Leu Arg GlnAla Lys Gln 465 470 475 480 Ser Ser Ser Ala Ala Leu Ser Lys Phe Ile AlaVal Tyr Phe Asp Tyr 485 490 495 Ser Cys Glu Gly Asp Val Pro Gly Ile LeuAsp Leu Ser Thr Lys Tyr 500 505 510 Arg Leu Met Asp Asn Leu Pro Gln LeuCys Ser His Leu His Ser Arg 515 520 525 Asp His Gly Leu Gln Glu Pro GlyGln His Thr Arg Gln Gly Ser Arg 530 535 540 Arg Asn Tyr Phe Arg Ser LysSer Gly Arg Ser Leu Tyr Val Ala Ile 545 550 555 560 Cys Asn Met His GlnPhe Ile Asp Glu Glu Pro Asp Trp Phe Glu Lys 565 570 575 Gln Phe Val ProPhe His Pro Pro Pro Leu Arg Tyr Arg Glu Pro Val 580 585 590 Leu Glu LysPhe Asp Ser Gly Leu Val Leu Asn Asp Val Met Cys Lys 595 600 605 Pro GlyPro Glu Ser Asp Phe Cys Leu Lys Val Glu Ala Ala Val Leu 610 615 620 GlyAla Thr Gly Pro Ala Asp Ser Gln His Glu Ser Gln His Gly Gly 625 630 635640 Leu Asp Gln Asp Gly Glu Ala Arg Pro Ala Leu Asp Gly Ser Ala Ala 645650 655 Leu Gln Pro Leu Leu His Thr Val Lys Ala Gly Ser Pro Ser Asp Met660 665 670 Pro Arg Asp Ser Gly Ile Tyr Asp Ser Ser Val Pro Ser Ser GluLeu 675 680 685 Ser Leu Pro Leu Met Glu Gly Leu Ser Thr Asp Gln Thr GluThr Ser 690 695 700 Ser Leu Thr Glu Ser Val Ser Ser Ser Ser Gly Leu GlyGlu Glu Glu 705 710 715 720 Pro Pro Ala Leu Pro Ser Lys Leu Leu Ser SerGly Ser Cys Lys Ala 725 730 735 Asp Leu Gly Cys Arg Ser Tyr Thr Asp GluLeu His Ala Val Ala Pro 740 745 750 Leu 3 2259 DNA Artificial SequenceThis degenerate nucleotide sequence encodes the amino acid sequence ofSEQ ID NO2. 3 atggcnccnt ggytncaryt ntgywsngtn ttyttyacng tnaaygcntgyytnaayggn 60 wsncarytng cngtngcngc nggnggnwsn ggnmgngcnm gnggngcngayacntgyggn 120 tggmgnatga argcngcngc nmgnccnmgn ytntgygtng cnaaygarggngtnggnccn 180 gcnwsnmgna aywsnggnyt ntayaayath acnttyaart aygayaaytgyacnacntay 240 ytnaayccng tnggnaarca ygtnathgcn gaygcncara ayathacnathwsncartay 300 gcntgycayg aycargtngc ngtnacnath ytntggwsnc cnggngcnytnggnathgar 360 ttyytnaarg gnttymgngt nathytngar garytnaarw sngarggnmgncartgycar 420 carytnathy tnaargaycc naarcarytn aaywsnwsnt tyaarmgnacnggnatggar 480 wsncarccnt tyytnaayat gaarttygar acngaytayt tygtnaargtngtnccntty 540 ccnwsnatha araaygarws naaytaycay ccnttyttyt tymgnacnmgngcntgygay 600 ytnytnytnc arccngayaa yytngcntgy aarccnttyt ggaarccnmgnaayytnaay 660 athwsncarc ayggnwsnga yatgcargtn wsnttygayc aygcnccncayaayttyggn 720 ttymgnttyt tytayytnca ytayaarytn aarcaygarg gnccnttyaarmgnaaracn 780 tgyaarcarg arcaracnac ngaracnacn wsntgyytny tncaraaygtnwsnccnggn 840 gaytayatha thgarytngt ngaygayacn aayacnacnm gnaargtnatgcaytaygcn 900 ytnaarccng tncaywsncc ntgggcnggn ccnathmgng cngtngcnathacngtnccn 960 ytngtngtna thwsngcntt ygcnacnytn ttyacngtna tgtgymgnaaraarcarcar 1020 garaayatht aywsncayyt ngaygargar wsnwsngarw snwsnacntayacngcngcn 1080 ytnccnmgng armgnytnmg nccnmgnccn aargtnttyy tntgytaywsnwsnaargay 1140 ggncaraayc ayatgaaygt ngtncartgy ttygcntayt tyytncargayttytgyggn 1200 tgygargtng cnytngayyt ntgggargay ttywsnytnt gymgngarggncarmgngar 1260 tgggtnathc araarathca ygarwsncar ttyathathg tngtntgywsnaarggnatg 1320 aartayttyg tngayaaraa raaytayaar cayaarggng gnggnmgnggnwsnggnaar 1380 ggngarytnt tyytngtngc ngtnwsngcn athgcngara arytnmgncargcnaarcar 1440 wsnwsnwsng cngcnytnws naarttyath gcngtntayt tygaytaywsntgygarggn 1500 gaygtnccng gnathytnga yytnwsnacn aartaymgny tnatggayaayytnccncar 1560 ytntgywsnc ayytncayws nmgngaycay ggnytncarg arccnggncarcayacnmgn 1620 carggnwsnm gnmgnaayta yttymgnwsn aarwsnggnm gnwsnytntaygtngcnath 1680 tgyaayatgc aycarttyat hgaygargar ccngaytggt tygaraarcarttygtnccn 1740 ttycayccnc cnccnytnmg ntaymgngar ccngtnytng araarttygaywsnggnytn 1800 gtnytnaayg aygtnatgtg yaarccnggn ccngarwsng ayttytgyytnaargtngar 1860 gcngcngtny tnggngcnac nggnccngcn gaywsncarc aygarwsncarcayggnggn 1920 ytngaycarg ayggngargc nmgnccngcn ytngayggnw sngcngcnytncarccnytn 1980 ytncayacng tnaargcngg nwsnccnwsn gayatgccnm gngaywsnggnathtaygay 2040 wsnwsngtnc cnwsnwsnga rytnwsnytn ccnytnatgg arggnytnwsnacngaycar 2100 acngaracnw snwsnytnac ngarwsngtn wsnwsnwsnw snggnytnggngargargar 2160 ccnccngcny tnccnwsnaa rytnytnwsn wsnggnwsnt gyaargcngayytnggntgy 2220 mgnwsntaya cngaygaryt ncaygcngtn gcnccnytn 2259 4 2383DNA Homo sapiens CDS (86)...(2344) 4 ccgccgcggc caccgcccac tcggggctggccagcggcgg gcggccgggg cgcagagaac 60 ggcctggctg ggcgagcgca cggcc atg gccccg tgg ctg cag ctc tgc tcc 112 Met Ala Pro Trp Leu Gln Leu Cys Ser 1 5gtc ttc ttt acg gtc aac gcc tgc ctc aac ggc tcg cag ctg gct gtg 160 ValPhe Phe Thr Val Asn Ala Cys Leu Asn Gly Ser Gln Leu Ala Val 10 15 20 25gcc gct ggc ggg tcc ggc cgc gcg cgg ggc gcc gac acc tgt ggc tgg 208 AlaAla Gly Gly Ser Gly Arg Ala Arg Gly Ala Asp Thr Cys Gly Trp 30 35 40 aggatg aaa gcg gct gcc cga ccc cgg ctt tgt gtt gct aat gag gga 256 Arg MetLys Ala Ala Ala Arg Pro Arg Leu Cys Val Ala Asn Glu Gly 45 50 55 gtg gggcca gcc agc aga aac agt ggg ctg tac aac atc acc ttc aaa 304 Val Gly ProAla Ser Arg Asn Ser Gly Leu Tyr Asn Ile Thr Phe Lys 60 65 70 tat gac aattgt acc acc tac ttg aat cca gtg ggg aag cat gtg att 352 Tyr Asp Asn CysThr Thr Tyr Leu Asn Pro Val Gly Lys His Val Ile 75 80 85 gct gac gcc cagaat atc acc atc agc cag tat gct tgc cat gac caa 400 Ala Asp Ala Gln AsnIle Thr Ile Ser Gln Tyr Ala Cys His Asp Gln 90 95 100 105 gtg gca gtcacc att ctt tgg tcc cca ggg gcc ctc ggc atc gaa ttc 448 Val Ala Val ThrIle Leu Trp Ser Pro Gly Ala Leu Gly Ile Glu Phe 110 115 120 ctg aaa ggattt cgg gta ata ctg gag gag ctg aag tcg gag gga aga 496 Leu Lys Gly PheArg Val Ile Leu Glu Glu Leu Lys Ser Glu Gly Arg 125 130 135 cag tgc caacaa ctg att cta aag gat ccg aag cag ctc aac agt agc 544 Gln Cys Gln GlnLeu Ile Leu Lys Asp Pro Lys Gln Leu Asn Ser Ser 140 145 150 ttc aaa agaact gga atg gaa tct caa cct ttc ctg aat atg aaa ttt 592 Phe Lys Arg ThrGly Met Glu Ser Gln Pro Phe Leu Asn Met Lys Phe 155 160 165 gaa acg gattat ttc gta aag gtt gtc cct ttt cct tcc att aaa aac 640 Glu Thr Asp TyrPhe Val Lys Val Val Pro Phe Pro Ser Ile Lys Asn 170 175 180 185 gaa agcaat tac cac cct ttc ttc ttt aga acc cga gcc tgt gac ctg 688 Glu Ser AsnTyr His Pro Phe Phe Phe Arg Thr Arg Ala Cys Asp Leu 190 195 200 ttg ttacag ccg gac aat cta gct tgt aaa ccc ttc tgg aag cct cgg 736 Leu Leu GlnPro Asp Asn Leu Ala Cys Lys Pro Phe Trp Lys Pro Arg 205 210 215 aac ctgaac atc agc cag cat ggc tcg gac atg cag gtg tcc ttc gac 784 Asn Leu AsnIle Ser Gln His Gly Ser Asp Met Gln Val Ser Phe Asp 220 225 230 cac gcaccg cac aac ttc ggc ttc cgt ttc ttc tat ctt cac tac aag 832 His Ala ProHis Asn Phe Gly Phe Arg Phe Phe Tyr Leu His Tyr Lys 235 240 245 ctc aagcac gaa gga cct ttc aag cga aag acc tgt aag cag gag caa 880 Leu Lys HisGlu Gly Pro Phe Lys Arg Lys Thr Cys Lys Gln Glu Gln 250 255 260 265 actaca gag atg acc agc tgc ctc ctt caa aat gtt tct cca ggg gat 928 Thr ThrGlu Met Thr Ser Cys Leu Leu Gln Asn Val Ser Pro Gly Asp 270 275 280 tatata att gag ctg gtg gat gac act aac aca aca aga aaa gtg atg 976 Tyr IleIle Glu Leu Val Asp Asp Thr Asn Thr Thr Arg Lys Val Met 285 290 295 cattat gcc tta aag cca gtg cac tcc ccg tgg gcc ggg ccc atc aga 1024 His TyrAla Leu Lys Pro Val His Ser Pro Trp Ala Gly Pro Ile Arg 300 305 310 gccgtg gcc atc aca gtg cca ctg gta gtc ata tcg gca ttc gcg acg 1072 Ala ValAla Ile Thr Val Pro Leu Val Val Ile Ser Ala Phe Ala Thr 315 320 325 ctcttc act gtg atg tgc cgc aag aag caa caa gaa aat ata tat tca 1120 Leu PheThr Val Met Cys Arg Lys Lys Gln Gln Glu Asn Ile Tyr Ser 330 335 340 345cat tta gat gaa gag agc tct gag tct tcc aca tac act gca gca ctc 1168 HisLeu Asp Glu Glu Ser Ser Glu Ser Ser Thr Tyr Thr Ala Ala Leu 350 355 360cca aga gag agg ctc cgg ccg cgg ccg aag gtc ttt ctc tgc tat tcc 1216 ProArg Glu Arg Leu Arg Pro Arg Pro Lys Val Phe Leu Cys Tyr Ser 365 370 375agt aaa gat ggc cag aat cac atg aat gtc gtc cag tgt ttc gcc tac 1264 SerLys Asp Gly Gln Asn His Met Asn Val Val Gln Cys Phe Ala Tyr 380 385 390ttc ctc cag gac ttc tgt ggc tgt gag gtg gct ctg gac ctg tgg gaa 1312 PheLeu Gln Asp Phe Cys Gly Cys Glu Val Ala Leu Asp Leu Trp Glu 395 400 405gac ttc agc ctc tgt aga gaa ggg cag aga gaa tgg gtc atc cag aag 1360 AspPhe Ser Leu Cys Arg Glu Gly Gln Arg Glu Trp Val Ile Gln Lys 410 415 420425 atc cac gag tcc cag ttc atc att gtg gtt tgt tcc aaa ggt atg aag 1408Ile His Glu Ser Gln Phe Ile Ile Val Val Cys Ser Lys Gly Met Lys 430 435440 tac ttt gtg gac aag aag aac tac aaa cac aaa gga ggt ggc cga ggc 1456Tyr Phe Val Asp Lys Lys Asn Tyr Lys His Lys Gly Gly Gly Arg Gly 445 450455 tcg ggg aaa gga gag ctc ttc ctg gtg gcg gtg tca gcc att gcc gaa 1504Ser Gly Lys Gly Glu Leu Phe Leu Val Ala Val Ser Ala Ile Ala Glu 460 465470 aag ctc cgc cag gcc aag cag agt tcg tcc gcg gcg ctc agc aag ttt 1552Lys Leu Arg Gln Ala Lys Gln Ser Ser Ser Ala Ala Leu Ser Lys Phe 475 480485 atc gcc gtc tac ttt gat tat tcc tgc gag gga gac gtc ccc ggt atc 1600Ile Ala Val Tyr Phe Asp Tyr Ser Cys Glu Gly Asp Val Pro Gly Ile 490 495500 505 cta gac ctg agt acc aag tac aga ctc atg gac aat ctt cct cag ctc1648 Leu Asp Leu Ser Thr Lys Tyr Arg Leu Met Asp Asn Leu Pro Gln Leu 510515 520 tgt tcc cac ctg cac tcc cga gac cac ggc ctc cag gag ccg ggg cag1696 Cys Ser His Leu His Ser Arg Asp His Gly Leu Gln Glu Pro Gly Gln 525530 535 cac acg cga cag ggc agc aga agg aac tac ttc cgg agc aag tca ggc1744 His Thr Arg Gln Gly Ser Arg Arg Asn Tyr Phe Arg Ser Lys Ser Gly 540545 550 cgg tcc cta tac gtc gcc att tgc aac atg cac cag ttt att gac gag1792 Arg Ser Leu Tyr Val Ala Ile Cys Asn Met His Gln Phe Ile Asp Glu 555560 565 gag ccc gac tgg ttc gaa aag cag ttc gtt ccc ttc cat cct cct cca1840 Glu Pro Asp Trp Phe Glu Lys Gln Phe Val Pro Phe His Pro Pro Pro 570575 580 585 ctg cgc tac cgg gag cca gtc ttg gag aaa ttt gat tcg ggc ttggtt 1888 Leu Arg Tyr Arg Glu Pro Val Leu Glu Lys Phe Asp Ser Gly Leu Val590 595 600 tta aat gat gtc atg tgc aaa cca ggg cct gag agt gac ttc tgccta 1936 Leu Asn Asp Val Met Cys Lys Pro Gly Pro Glu Ser Asp Phe Cys Leu605 610 615 aag gta gag gcg gct gtt ctt ggg gca acc gga cca gcc gac tcccag 1984 Lys Val Glu Ala Ala Val Leu Gly Ala Thr Gly Pro Ala Asp Ser Gln620 625 630 cac gag agt cag cat ggg ggc ctg gac caa gac ggg gag gcc cggcct 2032 His Glu Ser Gln His Gly Gly Leu Asp Gln Asp Gly Glu Ala Arg Pro635 640 645 gcc ctt gac ggt agc gcc gcc ctg caa ccc ctg ctg cac acg gtgaaa 2080 Ala Leu Asp Gly Ser Ala Ala Leu Gln Pro Leu Leu His Thr Val Lys650 655 660 665 gcc ggc agc ccc tcg gac atg ccg cgg gac tca ggc atc tatgac tcg 2128 Ala Gly Ser Pro Ser Asp Met Pro Arg Asp Ser Gly Ile Tyr AspSer 670 675 680 tct gtg ccc tca tcc gag ctg tct ctg cca ctg atg gaa ggactc tcg 2176 Ser Val Pro Ser Ser Glu Leu Ser Leu Pro Leu Met Glu Gly LeuSer 685 690 695 acg gac cag aca gaa acg tct tcc ctg acg gag agc gtg tcctcc tct 2224 Thr Asp Gln Thr Glu Thr Ser Ser Leu Thr Glu Ser Val Ser SerSer 700 705 710 tca ggc ctg ggt gag gag gaa cct cct gcc ctt cct tcc aagctc ctc 2272 Ser Gly Leu Gly Glu Glu Glu Pro Pro Ala Leu Pro Ser Lys LeuLeu 715 720 725 tct tct ggg tca tgc aaa gca gat ctt ggt tgc cgc agc tacact gat 2320 Ser Ser Gly Ser Cys Lys Ala Asp Leu Gly Cys Arg Ser Tyr ThrAsp 730 735 740 745 gaa ctc cac gcg gcc gcc cct ttg taacaaaacgaaagagtcta agcattgcca 2374 Glu Leu His Ala Ala Ala Pro Leu 750 ctttagctg2383 5 753 PRT Homo sapiens 5 Met Ala Pro Trp Leu Gln Leu Cys Ser ValPhe Phe Thr Val Asn Ala 1 5 10 15 Cys Leu Asn Gly Ser Gln Leu Ala ValAla Ala Gly Gly Ser Gly Arg 20 25 30 Ala Arg Gly Ala Asp Thr Cys Gly TrpArg Met Lys Ala Ala Ala Arg 35 40 45 Pro Arg Leu Cys Val Ala Asn Glu GlyVal Gly Pro Ala Ser Arg Asn 50 55 60 Ser Gly Leu Tyr Asn Ile Thr Phe LysTyr Asp Asn Cys Thr Thr Tyr 65 70 75 80 Leu Asn Pro Val Gly Lys His ValIle Ala Asp Ala Gln Asn Ile Thr 85 90 95 Ile Ser Gln Tyr Ala Cys His AspGln Val Ala Val Thr Ile Leu Trp 100 105 110 Ser Pro Gly Ala Leu Gly IleGlu Phe Leu Lys Gly Phe Arg Val Ile 115 120 125 Leu Glu Glu Leu Lys SerGlu Gly Arg Gln Cys Gln Gln Leu Ile Leu 130 135 140 Lys Asp Pro Lys GlnLeu Asn Ser Ser Phe Lys Arg Thr Gly Met Glu 145 150 155 160 Ser Gln ProPhe Leu Asn Met Lys Phe Glu Thr Asp Tyr Phe Val Lys 165 170 175 Val ValPro Phe Pro Ser Ile Lys Asn Glu Ser Asn Tyr His Pro Phe 180 185 190 PhePhe Arg Thr Arg Ala Cys Asp Leu Leu Leu Gln Pro Asp Asn Leu 195 200 205Ala Cys Lys Pro Phe Trp Lys Pro Arg Asn Leu Asn Ile Ser Gln His 210 215220 Gly Ser Asp Met Gln Val Ser Phe Asp His Ala Pro His Asn Phe Gly 225230 235 240 Phe Arg Phe Phe Tyr Leu His Tyr Lys Leu Lys His Glu Gly ProPhe 245 250 255 Lys Arg Lys Thr Cys Lys Gln Glu Gln Thr Thr Glu Met ThrSer Cys 260 265 270 Leu Leu Gln Asn Val Ser Pro Gly Asp Tyr Ile Ile GluLeu Val Asp 275 280 285 Asp Thr Asn Thr Thr Arg Lys Val Met His Tyr AlaLeu Lys Pro Val 290 295 300 His Ser Pro Trp Ala Gly Pro Ile Arg Ala ValAla Ile Thr Val Pro 305 310 315 320 Leu Val Val Ile Ser Ala Phe Ala ThrLeu Phe Thr Val Met Cys Arg 325 330 335 Lys Lys Gln Gln Glu Asn Ile TyrSer His Leu Asp Glu Glu Ser Ser 340 345 350 Glu Ser Ser Thr Tyr Thr AlaAla Leu Pro Arg Glu Arg Leu Arg Pro 355 360 365 Arg Pro Lys Val Phe LeuCys Tyr Ser Ser Lys Asp Gly Gln Asn His 370 375 380 Met Asn Val Val GlnCys Phe Ala Tyr Phe Leu Gln Asp Phe Cys Gly 385 390 395 400 Cys Glu ValAla Leu Asp Leu Trp Glu Asp Phe Ser Leu Cys Arg Glu 405 410 415 Gly GlnArg Glu Trp Val Ile Gln Lys Ile His Glu Ser Gln Phe Ile 420 425 430 IleVal Val Cys Ser Lys Gly Met Lys Tyr Phe Val Asp Lys Lys Asn 435 440 445Tyr Lys His Lys Gly Gly Gly Arg Gly Ser Gly Lys Gly Glu Leu Phe 450 455460 Leu Val Ala Val Ser Ala Ile Ala Glu Lys Leu Arg Gln Ala Lys Gln 465470 475 480 Ser Ser Ser Ala Ala Leu Ser Lys Phe Ile Ala Val Tyr Phe AspTyr 485 490 495 Ser Cys Glu Gly Asp Val Pro Gly Ile Leu Asp Leu Ser ThrLys Tyr 500 505 510 Arg Leu Met Asp Asn Leu Pro Gln Leu Cys Ser His LeuHis Ser Arg 515 520 525 Asp His Gly Leu Gln Glu Pro Gly Gln His Thr ArgGln Gly Ser Arg 530 535 540 Arg Asn Tyr Phe Arg Ser Lys Ser Gly Arg SerLeu Tyr Val Ala Ile 545 550 555 560 Cys Asn Met His Gln Phe Ile Asp GluGlu Pro Asp Trp Phe Glu Lys 565 570 575 Gln Phe Val Pro Phe His Pro ProPro Leu Arg Tyr Arg Glu Pro Val 580 585 590 Leu Glu Lys Phe Asp Ser GlyLeu Val Leu Asn Asp Val Met Cys Lys 595 600 605 Pro Gly Pro Glu Ser AspPhe Cys Leu Lys Val Glu Ala Ala Val Leu 610 615 620 Gly Ala Thr Gly ProAla Asp Ser Gln His Glu Ser Gln His Gly Gly 625 630 635 640 Leu Asp GlnAsp Gly Glu Ala Arg Pro Ala Leu Asp Gly Ser Ala Ala 645 650 655 Leu GlnPro Leu Leu His Thr Val Lys Ala Gly Ser Pro Ser Asp Met 660 665 670 ProArg Asp Ser Gly Ile Tyr Asp Ser Ser Val Pro Ser Ser Glu Leu 675 680 685Ser Leu Pro Leu Met Glu Gly Leu Ser Thr Asp Gln Thr Glu Thr Ser 690 695700 Ser Leu Thr Glu Ser Val Ser Ser Ser Ser Gly Leu Gly Glu Glu Glu 705710 715 720 Pro Pro Ala Leu Pro Ser Lys Leu Leu Ser Ser Gly Ser Cys LysAla 725 730 735 Asp Leu Gly Cys Arg Ser Tyr Thr Asp Glu Leu His Ala AlaAla Pro 740 745 750 Leu 6 2259 DNA Artificial Sequence This degeneratenucleotide sequence encodes the amino acid sequence of SEQ ID NO5. 6atggcnccnt ggytncaryt ntgywsngtn ttyttyacng tnaaygcntg yytnaayggn 60wsncarytng cngtngcngc nggnggnwsn ggnmgngcnm gnggngcnga yacntgyggn 120tggmgnatga argcngcngc nmgnccnmgn ytntgygtng cnaaygargg ngtnggnccn 180gcnwsnmgna aywsnggnyt ntayaayath acnttyaart aygayaaytg yacnacntay 240ytnaayccng tnggnaarca ygtnathgcn gaygcncara ayathacnat hwsncartay 300gcntgycayg aycargtngc ngtnacnath ytntggwsnc cnggngcnyt nggnathgar 360ttyytnaarg gnttymgngt nathytngar garytnaarw sngarggnmg ncartgycar 420carytnathy tnaargaycc naarcarytn aaywsnwsnt tyaarmgnac nggnatggar 480wsncarccnt tyytnaayat gaarttygar acngaytayt tygtnaargt ngtnccntty 540ccnwsnatha araaygarws naaytaycay ccnttyttyt tymgnacnmg ngcntgygay 600ytnytnytnc arccngayaa yytngcntgy aarccnttyt ggaarccnmg naayytnaay 660athwsncarc ayggnwsnga yatgcargtn wsnttygayc aygcnccnca yaayttyggn 720ttymgnttyt tytayytnca ytayaarytn aarcaygarg gnccnttyaa rmgnaaracn 780tgyaarcarg arcaracnac ngaratgacn wsntgyytny tncaraaygt nwsnccnggn 840gaytayatha thgarytngt ngaygayacn aayacnacnm gnaargtnat gcaytaygcn 900ytnaarccng tncaywsncc ntgggcnggn ccnathmgng cngtngcnat hacngtnccn 960ytngtngtna thwsngcntt ygcnacnytn ttyacngtna tgtgymgnaa raarcarcar 1020garaayatht aywsncayyt ngaygargar wsnwsngarw snwsnacnta yacngcngcn 1080ytnccnmgng armgnytnmg nccnmgnccn aargtnttyy tntgytayws nwsnaargay 1140ggncaraayc ayatgaaygt ngtncartgy ttygcntayt tyytncarga yttytgyggn 1200tgygargtng cnytngayyt ntgggargay ttywsnytnt gymgngargg ncarmgngar 1260tgggtnathc araarathca ygarwsncar ttyathathg tngtntgyws naarggnatg 1320aartayttyg tngayaaraa raaytayaar cayaarggng gnggnmgngg nwsnggnaar 1380ggngarytnt tyytngtngc ngtnwsngcn athgcngara arytnmgnca rgcnaarcar 1440wsnwsnwsng cngcnytnws naarttyath gcngtntayt tygaytayws ntgygarggn 1500gaygtnccng gnathytnga yytnwsnacn aartaymgny tnatggayaa yytnccncar 1560ytntgywsnc ayytncayws nmgngaycay ggnytncarg arccnggnca rcayacnmgn 1620carggnwsnm gnmgnaayta yttymgnwsn aarwsnggnm gnwsnytnta ygtngcnath 1680tgyaayatgc aycarttyat hgaygargar ccngaytggt tygaraarca rttygtnccn 1740ttycayccnc cnccnytnmg ntaymgngar ccngtnytng araarttyga ywsnggnytn 1800gtnytnaayg aygtnatgtg yaarccnggn ccngarwsng ayttytgyyt naargtngar 1860gcngcngtny tnggngcnac nggnccngcn gaywsncarc aygarwsnca rcayggnggn 1920ytngaycarg ayggngargc nmgnccngcn ytngayggnw sngcngcnyt ncarccnytn 1980ytncayacng tnaargcngg nwsnccnwsn gayatgccnm gngaywsngg nathtaygay 2040wsnwsngtnc cnwsnwsnga rytnwsnytn ccnytnatgg arggnytnws nacngaycar 2100acngaracnw snwsnytnac ngarwsngtn wsnwsnwsnw snggnytngg ngargargar 2160ccnccngcny tnccnwsnaa rytnytnwsn wsnggnwsnt gyaargcnga yytnggntgy 2220mgnwsntaya cngaygaryt ncaygcngcn gcnccnytn 2259 7 2341 DNA Homo sapiensCDS (86)...(2302) 7 ccgccgcggc caccgcccac tcggggctgg ccagcggcgggcggccgggg cgcagagaac 60 ggcctggctg ggcgagcgca cggcc atg gcc ccg tgg ctgcag ctc tgc tcc 112 Met Ala Pro Trp Leu Gln Leu Cys Ser 1 5 gtc ttc tttacg gtc aac gcc tgc ctc aac ggc tcg cag ctg gct gtg 160 Val Phe Phe ThrVal Asn Ala Cys Leu Asn Gly Ser Gln Leu Ala Val 10 15 20 25 gcc gct ggcggg tcc ggc cgc gcg cgg ggc gcc gac acc tgt ggc tgg 208 Ala Ala Gly GlySer Gly Arg Ala Arg Gly Ala Asp Thr Cys Gly Trp 30 35 40 agg gga gtg gggcca gcc agc aga aac agt ggg ctg tac aac atc acc 256 Arg Gly Val Gly ProAla Ser Arg Asn Ser Gly Leu Tyr Asn Ile Thr 45 50 55 ttc aaa tat gac aattgt acc acc tac ttg aat cca gtg ggg aag cat 304 Phe Lys Tyr Asp Asn CysThr Thr Tyr Leu Asn Pro Val Gly Lys His 60 65 70 gtg att gct gac gcc cagaat atc acc atc agc cag tat gct tgc cat 352 Val Ile Ala Asp Ala Gln AsnIle Thr Ile Ser Gln Tyr Ala Cys His 75 80 85 gac caa gtg gca gtc acc attctt tgg tcc cca ggg gcc ctc ggc atc 400 Asp Gln Val Ala Val Thr Ile LeuTrp Ser Pro Gly Ala Leu Gly Ile 90 95 100 105 gaa ttc ctg aaa gga tttcgg gta ata ctg gag gag ctg aag tcg gag 448 Glu Phe Leu Lys Gly Phe ArgVal Ile Leu Glu Glu Leu Lys Ser Glu 110 115 120 gga aga cag tgc caa caactg att cta aag gat ccg aag cag ctc aac 496 Gly Arg Gln Cys Gln Gln LeuIle Leu Lys Asp Pro Lys Gln Leu Asn 125 130 135 agt agc ttc aaa aga actgga atg gaa tct caa cct ttc ctg aat atg 544 Ser Ser Phe Lys Arg Thr GlyMet Glu Ser Gln Pro Phe Leu Asn Met 140 145 150 aaa ttt gaa acg gat tatttc gta aag gtt gtc cct ttt cct tcc att 592 Lys Phe Glu Thr Asp Tyr PheVal Lys Val Val Pro Phe Pro Ser Ile 155 160 165 aaa aac gaa agc aat taccac cct ttc ttc ttt aga acc cga gcc tgt 640 Lys Asn Glu Ser Asn Tyr HisPro Phe Phe Phe Arg Thr Arg Ala Cys 170 175 180 185 gac ctg ttg tta cagccg gac aat cta gct tgt aaa ccc ttc tgg aag 688 Asp Leu Leu Leu Gln ProAsp Asn Leu Ala Cys Lys Pro Phe Trp Lys 190 195 200 cct cgg aac ctg aacatc agc cag cat ggc tcg gac atg cag gtg tcc 736 Pro Arg Asn Leu Asn IleSer Gln His Gly Ser Asp Met Gln Val Ser 205 210 215 ttc gac cat gca ccgcac aac ttc ggc ttc cgt ttc ttc tat ctt cac 784 Phe Asp His Ala Pro HisAsn Phe Gly Phe Arg Phe Phe Tyr Leu His 220 225 230 tac aag ctc aag cacgaa gga cct ttc aag cga aag acc tgt aag cag 832 Tyr Lys Leu Lys His GluGly Pro Phe Lys Arg Lys Thr Cys Lys Gln 235 240 245 gag caa act aca gagacg acc agc tgc ctc ctt caa aat gtt tct cca 880 Glu Gln Thr Thr Glu ThrThr Ser Cys Leu Leu Gln Asn Val Ser Pro 250 255 260 265 ggg gat tat ataatt gag ctg gtg gat gac act aac aca aca aga aaa 928 Gly Asp Tyr Ile IleGlu Leu Val Asp Asp Thr Asn Thr Thr Arg Lys 270 275 280 gtg atg cat tatgcc tta aag cca gtg cac tcc ccg tgg gcc ggg ccc 976 Val Met His Tyr AlaLeu Lys Pro Val His Ser Pro Trp Ala Gly Pro 285 290 295 atc aga gcc gtggcc atc aca gtg cca ctg gta gtc ata tcg gca ttc 1024 Ile Arg Ala Val AlaIle Thr Val Pro Leu Val Val Ile Ser Ala Phe 300 305 310 gcg acg ctc ttcact gtg atg tgc cgc aag aag caa caa gaa aat ata 1072 Ala Thr Leu Phe ThrVal Met Cys Arg Lys Lys Gln Gln Glu Asn Ile 315 320 325 tat tca cat ttagat gaa gag agc tct gag tct tcc aca tac act gca 1120 Tyr Ser His Leu AspGlu Glu Ser Ser Glu Ser Ser Thr Tyr Thr Ala 330 335 340 345 gca ctc ccaaga gag agg ctc cgg ccg cgg ccg aag gtc ttt ctc tgc 1168 Ala Leu Pro ArgGlu Arg Leu Arg Pro Arg Pro Lys Val Phe Leu Cys 350 355 360 tat tcc agtaaa gat ggc cag aat cac atg aat gtc gtc cag tgt ttc 1216 Tyr Ser Ser LysAsp Gly Gln Asn His Met Asn Val Val Gln Cys Phe 365 370 375 gcc tac ttcctc cag gac ttc tgt ggc tgt gag gtg gct ctg gac ctg 1264 Ala Tyr Phe LeuGln Asp Phe Cys Gly Cys Glu Val Ala Leu Asp Leu 380 385 390 tgg gaa gacttc agc ctc tgt aga gaa ggg cag aga gaa tgg gtc atc 1312 Trp Glu Asp PheSer Leu Cys Arg Glu Gly Gln Arg Glu Trp Val Ile 395 400 405 cag aag atccac gag tcc cag ttc atc att gtg gtt tgt tcc aaa ggt 1360 Gln Lys Ile HisGlu Ser Gln Phe Ile Ile Val Val Cys Ser Lys Gly 410 415 420 425 atg aagtac ttt gtg gac aag aag aac tac aaa cac aaa gga ggt ggc 1408 Met Lys TyrPhe Val Asp Lys Lys Asn Tyr Lys His Lys Gly Gly Gly 430 435 440 cga ggctcg ggg aaa gga gag ctc ttc ctg gtg gcg gtg tca gcc att 1456 Arg Gly SerGly Lys Gly Glu Leu Phe Leu Val Ala Val Ser Ala Ile 445 450 455 gcc gaaaag ctc cgc cag gcc aag cag agt tcg tcc gcg gcg ctc agc 1504 Ala Glu LysLeu Arg Gln Ala Lys Gln Ser Ser Ser Ala Ala Leu Ser 460 465 470 aag tttatc gcc gtc tac ttt gat tat tcc tgc gag gga gac gtc ccc 1552 Lys Phe IleAla Val Tyr Phe Asp Tyr Ser Cys Glu Gly Asp Val Pro 475 480 485 ggt atccta gac ctg agt acc aag tac aga ctc atg gac aat ctt cct 1600 Gly Ile LeuAsp Leu Ser Thr Lys Tyr Arg Leu Met Asp Asn Leu Pro 490 495 500 505 cagctc tgt tcc cac ttg cac tcc cga gac cac ggc ctc cag gag ccg 1648 Gln LeuCys Ser His Leu His Ser Arg Asp His Gly Leu Gln Glu Pro 510 515 520 gggcag cac acg cga cag ggc agc aga agg aac tac ttc cgg agc aag 1696 Gly GlnHis Thr Arg Gln Gly Ser Arg Arg Asn Tyr Phe Arg Ser Lys 525 530 535 tcaggc cgg tcc cta tac gtc gcc att tgc aac atg cac cag ttt att 1744 Ser GlyArg Ser Leu Tyr Val Ala Ile Cys Asn Met His Gln Phe Ile 540 545 550 gacgag gag ccc gac tgg ttc gaa aag cag ttc gtt ccc ttc cat cct 1792 Asp GluGlu Pro Asp Trp Phe Glu Lys Gln Phe Val Pro Phe His Pro 555 560 565 cctcca ctg cgc tac cgg gag cca gtc ttg gag aaa ttt gat tcg ggc 1840 Pro ProLeu Arg Tyr Arg Glu Pro Val Leu Glu Lys Phe Asp Ser Gly 570 575 580 585ttg gtt tta aat gat gtc atg tgc aaa cca ggg cct gag agt gac ttc 1888 LeuVal Leu Asn Asp Val Met Cys Lys Pro Gly Pro Glu Ser Asp Phe 590 595 600tgc cta aag gta gag gcg gct gtt ctt ggg gca acc gga cca gcc gac 1936 CysLeu Lys Val Glu Ala Ala Val Leu Gly Ala Thr Gly Pro Ala Asp 605 610 615tcc cag cac gag agt cag cat ggg ggc ctg gac caa gac ggg gag gcc 1984 SerGln His Glu Ser Gln His Gly Gly Leu Asp Gln Asp Gly Glu Ala 620 625 630cgg cct gcc ctt gac ggt agc gcc gcc ctg caa ccc ctg ctg cac acg 2032 ArgPro Ala Leu Asp Gly Ser Ala Ala Leu Gln Pro Leu Leu His Thr 635 640 645gtg aaa gcc ggc agc ccc tcg gac atg ccg cgg gac tca ggc atc tat 2080 ValLys Ala Gly Ser Pro Ser Asp Met Pro Arg Asp Ser Gly Ile Tyr 650 655 660665 gac tcg tct gtg ccc tca tcc gag ctg tct ctg cca ctg atg gaa gga 2128Asp Ser Ser Val Pro Ser Ser Glu Leu Ser Leu Pro Leu Met Glu Gly 670 675680 ctc tcg acg gac cag aca gaa acg tct tcc ctg acg gag agc gtg tcc 2176Leu Ser Thr Asp Gln Thr Glu Thr Ser Ser Leu Thr Glu Ser Val Ser 685 690695 tcc tct tca ggc ctg ggt gag gag gaa cct cct gcc ctt cct tcc aag 2224Ser Ser Ser Gly Leu Gly Glu Glu Glu Pro Pro Ala Leu Pro Ser Lys 700 705710 ctc ctc tct tct ggg tca tgc aaa gca gat ctt ggt tgc cgc agc tac 2272Leu Leu Ser Ser Gly Ser Cys Lys Ala Asp Leu Gly Cys Arg Ser Tyr 715 720725 act gat gaa ctc cac gcg gtc gcc cct ttg taacaaaacg aaagagtcta 2322Thr Asp Glu Leu His Ala Val Ala Pro Leu 730 735 agcattgcca ctttagctg2341 8 739 PRT Homo sapiens 8 Met Ala Pro Trp Leu Gln Leu Cys Ser ValPhe Phe Thr Val Asn Ala 1 5 10 15 Cys Leu Asn Gly Ser Gln Leu Ala ValAla Ala Gly Gly Ser Gly Arg 20 25 30 Ala Arg Gly Ala Asp Thr Cys Gly TrpArg Gly Val Gly Pro Ala Ser 35 40 45 Arg Asn Ser Gly Leu Tyr Asn Ile ThrPhe Lys Tyr Asp Asn Cys Thr 50 55 60 Thr Tyr Leu Asn Pro Val Gly Lys HisVal Ile Ala Asp Ala Gln Asn 65 70 75 80 Ile Thr Ile Ser Gln Tyr Ala CysHis Asp Gln Val Ala Val Thr Ile 85 90 95 Leu Trp Ser Pro Gly Ala Leu GlyIle Glu Phe Leu Lys Gly Phe Arg 100 105 110 Val Ile Leu Glu Glu Leu LysSer Glu Gly Arg Gln Cys Gln Gln Leu 115 120 125 Ile Leu Lys Asp Pro LysGln Leu Asn Ser Ser Phe Lys Arg Thr Gly 130 135 140 Met Glu Ser Gln ProPhe Leu Asn Met Lys Phe Glu Thr Asp Tyr Phe 145 150 155 160 Val Lys ValVal Pro Phe Pro Ser Ile Lys Asn Glu Ser Asn Tyr His 165 170 175 Pro PhePhe Phe Arg Thr Arg Ala Cys Asp Leu Leu Leu Gln Pro Asp 180 185 190 AsnLeu Ala Cys Lys Pro Phe Trp Lys Pro Arg Asn Leu Asn Ile Ser 195 200 205Gln His Gly Ser Asp Met Gln Val Ser Phe Asp His Ala Pro His Asn 210 215220 Phe Gly Phe Arg Phe Phe Tyr Leu His Tyr Lys Leu Lys His Glu Gly 225230 235 240 Pro Phe Lys Arg Lys Thr Cys Lys Gln Glu Gln Thr Thr Glu ThrThr 245 250 255 Ser Cys Leu Leu Gln Asn Val Ser Pro Gly Asp Tyr Ile IleGlu Leu 260 265 270 Val Asp Asp Thr Asn Thr Thr Arg Lys Val Met His TyrAla Leu Lys 275 280 285 Pro Val His Ser Pro Trp Ala Gly Pro Ile Arg AlaVal Ala Ile Thr 290 295 300 Val Pro Leu Val Val Ile Ser Ala Phe Ala ThrLeu Phe Thr Val Met 305 310 315 320 Cys Arg Lys Lys Gln Gln Glu Asn IleTyr Ser His Leu Asp Glu Glu 325 330 335 Ser Ser Glu Ser Ser Thr Tyr ThrAla Ala Leu Pro Arg Glu Arg Leu 340 345 350 Arg Pro Arg Pro Lys Val PheLeu Cys Tyr Ser Ser Lys Asp Gly Gln 355 360 365 Asn His Met Asn Val ValGln Cys Phe Ala Tyr Phe Leu Gln Asp Phe 370 375 380 Cys Gly Cys Glu ValAla Leu Asp Leu Trp Glu Asp Phe Ser Leu Cys 385 390 395 400 Arg Glu GlyGln Arg Glu Trp Val Ile Gln Lys Ile His Glu Ser Gln 405 410 415 Phe IleIle Val Val Cys Ser Lys Gly Met Lys Tyr Phe Val Asp Lys 420 425 430 LysAsn Tyr Lys His Lys Gly Gly Gly Arg Gly Ser Gly Lys Gly Glu 435 440 445Leu Phe Leu Val Ala Val Ser Ala Ile Ala Glu Lys Leu Arg Gln Ala 450 455460 Lys Gln Ser Ser Ser Ala Ala Leu Ser Lys Phe Ile Ala Val Tyr Phe 465470 475 480 Asp Tyr Ser Cys Glu Gly Asp Val Pro Gly Ile Leu Asp Leu SerThr 485 490 495 Lys Tyr Arg Leu Met Asp Asn Leu Pro Gln Leu Cys Ser HisLeu His 500 505 510 Ser Arg Asp His Gly Leu Gln Glu Pro Gly Gln His ThrArg Gln Gly 515 520 525 Ser Arg Arg Asn Tyr Phe Arg Ser Lys Ser Gly ArgSer Leu Tyr Val 530 535 540 Ala Ile Cys Asn Met His Gln Phe Ile Asp GluGlu Pro Asp Trp Phe 545 550 555 560 Glu Lys Gln Phe Val Pro Phe His ProPro Pro Leu Arg Tyr Arg Glu 565 570 575 Pro Val Leu Glu Lys Phe Asp SerGly Leu Val Leu Asn Asp Val Met 580 585 590 Cys Lys Pro Gly Pro Glu SerAsp Phe Cys Leu Lys Val Glu Ala Ala 595 600 605 Val Leu Gly Ala Thr GlyPro Ala Asp Ser Gln His Glu Ser Gln His 610 615 620 Gly Gly Leu Asp GlnAsp Gly Glu Ala Arg Pro Ala Leu Asp Gly Ser 625 630 635 640 Ala Ala LeuGln Pro Leu Leu His Thr Val Lys Ala Gly Ser Pro Ser 645 650 655 Asp MetPro Arg Asp Ser Gly Ile Tyr Asp Ser Ser Val Pro Ser Ser 660 665 670 GluLeu Ser Leu Pro Leu Met Glu Gly Leu Ser Thr Asp Gln Thr Glu 675 680 685Thr Ser Ser Leu Thr Glu Ser Val Ser Ser Ser Ser Gly Leu Gly Glu 690 695700 Glu Glu Pro Pro Ala Leu Pro Ser Lys Leu Leu Ser Ser Gly Ser Cys 705710 715 720 Lys Ala Asp Leu Gly Cys Arg Ser Tyr Thr Asp Glu Leu His AlaVal 725 730 735 Ala Pro Leu 9 2217 DNA Artificial Sequence Thisdegenerate nucleotide sequence encodes the amino acid sequence of SEQ IDNO8. 9 atggcnccnt ggytncaryt ntgywsngtn ttyttyacng tnaaygcntg yytnaayggn60 wsncarytng cngtngcngc nggnggnwsn ggnmgngcnm gnggngcnga yacntgyggn 120tggmgnggng tnggnccngc nwsnmgnaay wsnggnytnt ayaayathac nttyaartay 180gayaaytgya cnacntayyt naayccngtn ggnaarcayg tnathgcnga ygcncaraay 240athacnathw sncartaygc ntgycaygay cargtngcng tnacnathyt ntggwsnccn 300ggngcnytng gnathgartt yytnaarggn ttymgngtna thytngarga rytnaarwsn 360garggnmgnc artgycarca rytnathytn aargayccna arcarytnaa ywsnwsntty 420aarmgnacng gnatggarws ncarccntty ytnaayatga arttygarac ngaytaytty 480gtnaargtng tnccnttycc nwsnathaar aaygarwsna aytaycaycc nttyttytty 540mgnacnmgng cntgygayyt nytnytncar ccngayaayy tngcntgyaa rccnttytgg 600aarccnmgna ayytnaayat hwsncarcay ggnwsngaya tgcargtnws nttygaycay 660gcnccncaya ayttyggntt ymgnttytty tayytncayt ayaarytnaa rcaygarggn 720ccnttyaarm gnaaracntg yaarcargar caracnacng aracnacnws ntgyytnytn 780caraaygtnw snccnggnga ytayathath garytngtng aygayacnaa yacnacnmgn 840aargtnatgc aytaygcnyt naarccngtn caywsnccnt gggcnggncc nathmgngcn 900gtngcnatha cngtnccnyt ngtngtnath wsngcnttyg cnacnytntt yacngtnatg 960tgymgnaara arcarcarga raayathtay wsncayytng aygargarws nwsngarwsn 1020wsnacntaya cngcngcnyt nccnmgngar mgnytnmgnc cnmgnccnaa rgtnttyytn 1080tgytaywsnw snaargaygg ncaraaycay atgaaygtng tncartgytt ygcntaytty 1140ytncargayt tytgyggntg ygargtngcn ytngayytnt gggargaytt ywsnytntgy 1200mgngarggnc armgngartg ggtnathcar aarathcayg arwsncartt yathathgtn 1260gtntgywsna arggnatgaa rtayttygtn gayaaraara aytayaarca yaarggnggn 1320ggnmgnggnw snggnaargg ngarytntty ytngtngcng tnwsngcnat hgcngaraar 1380ytnmgncarg cnaarcarws nwsnwsngcn gcnytnwsna arttyathgc ngtntaytty 1440gaytaywsnt gygarggnga ygtnccnggn athytngayy tnwsnacnaa rtaymgnytn 1500atggayaayy tnccncaryt ntgywsncay ytncaywsnm gngaycaygg nytncargar 1560ccnggncarc ayacnmgnca rggnwsnmgn mgnaaytayt tymgnwsnaa rwsnggnmgn 1620wsnytntayg tngcnathtg yaayatgcay carttyathg aygargarcc ngaytggtty 1680garaarcart tygtnccntt ycayccnccn ccnytnmgnt aymgngarcc ngtnytngar 1740aarttygayw snggnytngt nytnaaygay gtnatgtgya arccnggncc ngarwsngay 1800ttytgyytna argtngargc ngcngtnytn ggngcnacng gnccngcnga ywsncarcay 1860garwsncarc ayggnggnyt ngaycargay ggngargcnm gnccngcnyt ngayggnwsn 1920gcngcnytnc arccnytnyt ncayacngtn aargcnggnw snccnwsnga yatgccnmgn 1980gaywsnggna thtaygayws nwsngtnccn wsnwsngary tnwsnytncc nytnatggar 2040ggnytnwsna cngaycarac ngaracnwsn wsnytnacng arwsngtnws nwsnwsnwsn 2100ggnytnggng argargarcc nccngcnytn ccnwsnaary tnytnwsnws nggnwsntgy 2160aargcngayy tnggntgymg nwsntayacn gaygarytnc aygcngtngc nccnytn 2217 1016 PRT Artificial Sequence Peptide linker. 10 Gly Gly Ser Gly Gly SerGly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 11 2443 DNA Mouse CDS(101)...(2317) 11 ctcggccgcc gccgctacca ccgccgccca ctcgggacta gagagcgagctacaggcagc 60 aacctagcgg agaccggccc aactgggcga gcgtacggcc atg gcc ccgtgg ctg 115 Met Ala Pro Trp Leu 1 5 cag ctc tgc tcc ttc ttc ttc act gtcaac gcc tgt ctc aac ggc tcg 163 Gln Leu Cys Ser Phe Phe Phe Thr Val AsnAla Cys Leu Asn Gly Ser 10 15 20 cag ctg gca gtg gcc gcg ggc ggc tcc ggccgc gcg agg ggc gcg gac 211 Gln Leu Ala Val Ala Ala Gly Gly Ser Gly ArgAla Arg Gly Ala Asp 25 30 35 acc tgt ggc tgg agg gga gtg ggg ccg gcc agcagg aac agc gga ctg 259 Thr Cys Gly Trp Arg Gly Val Gly Pro Ala Ser ArgAsn Ser Gly Leu 40 45 50 cac aac atc acc ttc aga tac gac aac tgt acc acctac ttg aat ccc 307 His Asn Ile Thr Phe Arg Tyr Asp Asn Cys Thr Thr TyrLeu Asn Pro 55 60 65 ggc ggc ggg aag cat gcg att gct gat gct cag aac atcacc atc agc 355 Gly Gly Gly Lys His Ala Ile Ala Asp Ala Gln Asn Ile ThrIle Ser 70 75 80 85 cag tac gct tgc cac gac cag gtg gca gtc acc att ctttgg tcc cca 403 Gln Tyr Ala Cys His Asp Gln Val Ala Val Thr Ile Leu TrpSer Pro 90 95 100 ggg gcc ctt ggc att gaa ttc cta aaa gga ttc cga gtcatc ctg gag 451 Gly Ala Leu Gly Ile Glu Phe Leu Lys Gly Phe Arg Val IleLeu Glu 105 110 115 gag ctg aag tcg gag ggc aga cag tgc caa cag ctg attcta aag gac 499 Glu Leu Lys Ser Glu Gly Arg Gln Cys Gln Gln Leu Ile LeuLys Asp 120 125 130 ccc aaa cag ctc aac agc agc ttc aga agg act gga atggaa tct cag 547 Pro Lys Gln Leu Asn Ser Ser Phe Arg Arg Thr Gly Met GluSer Gln 135 140 145 cct ttc ctg aat atg aaa ttt gag acg gat tac ttt gtaaag att gtc 595 Pro Phe Leu Asn Met Lys Phe Glu Thr Asp Tyr Phe Val LysIle Val 150 155 160 165 cct ttc cct tcc att aaa aat gaa agc aat tac catccc ttc ttc ttc 643 Pro Phe Pro Ser Ile Lys Asn Glu Ser Asn Tyr His ProPhe Phe Phe 170 175 180 aga aca cgg gcc tgt gac ctg ttg tta caa cct gacaac ttg gcc tgt 691 Arg Thr Arg Ala Cys Asp Leu Leu Leu Gln Pro Asp AsnLeu Ala Cys 185 190 195 aag cct ttc tgg aag cct cga aac ctg aat atc agccag cat ggt tct 739 Lys Pro Phe Trp Lys Pro Arg Asn Leu Asn Ile Ser GlnHis Gly Ser 200 205 210 gac atg cac gtg tcc ttc gac cat gcc ccg cag aacttc ggc ttc cgt 787 Asp Met His Val Ser Phe Asp His Ala Pro Gln Asn PheGly Phe Arg 215 220 225 ggc ttc cat gtt ctc tat aag ctc aag cac gaa ggcccc ttc agg cgg 835 Gly Phe His Val Leu Tyr Lys Leu Lys His Glu Gly ProPhe Arg Arg 230 235 240 245 agg act tgc agg cag gac cag aat aca gag acaacc agc tgc ctc ctc 883 Arg Thr Cys Arg Gln Asp Gln Asn Thr Glu Thr ThrSer Cys Leu Leu 250 255 260 caa aac gtt tct cca ggg gac tat atc att gagctg gtg gat gac agc 931 Gln Asn Val Ser Pro Gly Asp Tyr Ile Ile Glu LeuVal Asp Asp Ser 265 270 275 aac acc acc agg aaa gct gct cag tat gtg gtgaag tca gtg cag tct 979 Asn Thr Thr Arg Lys Ala Ala Gln Tyr Val Val LysSer Val Gln Ser 280 285 290 ccc tgg gct gga ccc atc aga gct gtg gcc atcact gtg cct ctg gtt 1027 Pro Trp Ala Gly Pro Ile Arg Ala Val Ala Ile ThrVal Pro Leu Val 295 300 305 gtc ata tct gcg ttc gca acc ctg ttc act gtgatg tgc aga aag aag 1075 Val Ile Ser Ala Phe Ala Thr Leu Phe Thr Val MetCys Arg Lys Lys 310 315 320 325 caa caa gaa aat ata tat tca cat tta gatgaa gaa agc ccg gag tcg 1123 Gln Gln Glu Asn Ile Tyr Ser His Leu Asp GluGlu Ser Pro Glu Ser 330 335 340 tcc aca tac gct gcg gct ctc ccc aga gacagg ctc cgg cct cag ccc 1171 Ser Thr Tyr Ala Ala Ala Leu Pro Arg Asp ArgLeu Arg Pro Gln Pro 345 350 355 aag gtc ttc ctc tgc tac tcc aat aaa gatggc cag aat cac atg aac 1219 Lys Val Phe Leu Cys Tyr Ser Asn Lys Asp GlyGln Asn His Met Asn 360 365 370 gtg gtc cag tgt ttc gcc tat ttc ctg caagat ttc tgt ggc tgt gag 1267 Val Val Gln Cys Phe Ala Tyr Phe Leu Gln AspPhe Cys Gly Cys Glu 375 380 385 gtg gct ctg gac ttg tgg gaa gat ttc agcctc tgc aga gag ggg cag 1315 Val Ala Leu Asp Leu Trp Glu Asp Phe Ser LeuCys Arg Glu Gly Gln 390 395 400 405 aga gaa tgg gcc att cag aag atc cacgag tcc cag ttc atc att gtc 1363 Arg Glu Trp Ala Ile Gln Lys Ile His GluSer Gln Phe Ile Ile Val 410 415 420 gtg tgc tcc aaa ggc atg aag tac tttgta gat aag aag aac ttc aga 1411 Val Cys Ser Lys Gly Met Lys Tyr Phe ValAsp Lys Lys Asn Phe Arg 425 430 435 cac aaa gga ggc agc cgc ggc gag gcgcaa ggc gag ttc ttc ctg gtg 1459 His Lys Gly Gly Ser Arg Gly Glu Ala GlnGly Glu Phe Phe Leu Val 440 445 450 gcc gtg gca gcc att gct gag aag ctccgt cag gcc aag cag agc tca 1507 Ala Val Ala Ala Ile Ala Glu Lys Leu ArgGln Ala Lys Gln Ser Ser 455 460 465 tct gcc gca ctg cgc aag ttc atc gccgtc tac ttc gat tat tcc tgt 1555 Ser Ala Ala Leu Arg Lys Phe Ile Ala ValTyr Phe Asp Tyr Ser Cys 470 475 480 485 gaa ggg gat gta ccc tgc agc ctggac ctg agc acc aag tac aag ctc 1603 Glu Gly Asp Val Pro Cys Ser Leu AspLeu Ser Thr Lys Tyr Lys Leu 490 495 500 atg gac cac ctt cct gag ctc tgtgcc cat ctg cac tca gga gag cag 1651 Met Asp His Leu Pro Glu Leu Cys AlaHis Leu His Ser Gly Glu Gln 505 510 515 gag gtg ctg ggt cag cac cca ggccac agc agc aga agg aac tac ttc 1699 Glu Val Leu Gly Gln His Pro Gly HisSer Ser Arg Arg Asn Tyr Phe 520 525 530 cgg agc aaa tcg ggc cgc tcc ctgtat gtt gcc att tgc aac atg cac 1747 Arg Ser Lys Ser Gly Arg Ser Leu TyrVal Ala Ile Cys Asn Met His 535 540 545 cag ttt att gat gag gag cct gactgg ttt gag aag cag ttt ata ccc 1795 Gln Phe Ile Asp Glu Glu Pro Asp TrpPhe Glu Lys Gln Phe Ile Pro 550 555 560 565 ttc caa cat ccc cct gtg cgctac cag gag cca gtc ctg gag aaa ttt 1843 Phe Gln His Pro Pro Val Arg TyrGln Glu Pro Val Leu Glu Lys Phe 570 575 580 gac tca ggc ttg gtt tta aatgat gtc ata agc aaa cca ggg cca gag 1891 Asp Ser Gly Leu Val Leu Asn AspVal Ile Ser Lys Pro Gly Pro Glu 585 590 595 agt gac ttc tgt cgg aaa gtcgag gct tgt gta ctt ggg gcc gct ggg 1939 Ser Asp Phe Cys Arg Lys Val GluAla Cys Val Leu Gly Ala Ala Gly 600 605 610 cca gcc gac tct tat tca tacctg gag agt cag cat gta ggc ctg gac 1987 Pro Ala Asp Ser Tyr Ser Tyr LeuGlu Ser Gln His Val Gly Leu Asp 615 620 625 caa gac act gag gcc cag ccctcc tgt gat agt gcc cct gcc ttg cag 2035 Gln Asp Thr Glu Ala Gln Pro SerCys Asp Ser Ala Pro Ala Leu Gln 630 635 640 645 ccc ctg tta cac gca gtgaaa gct ggc agt ccc tca gag atg cca cgg 2083 Pro Leu Leu His Ala Val LysAla Gly Ser Pro Ser Glu Met Pro Arg 650 655 660 gac tca ggc ata tat gattct tct gta ccc tca tca gag ctc tct ctg 2131 Asp Ser Gly Ile Tyr Asp SerSer Val Pro Ser Ser Glu Leu Ser Leu 665 670 675 cct ctg atg gag gga ctctcc ccg gat cag ata gaa aca tct tct ctg 2179 Pro Leu Met Glu Gly Leu SerPro Asp Gln Ile Glu Thr Ser Ser Leu 680 685 690 acc gag agt gta tct tcctcc tct ggc cta ggt gag gag gac ccc cct 2227 Thr Glu Ser Val Ser Ser SerSer Gly Leu Gly Glu Glu Asp Pro Pro 695 700 705 acc ctc cct tcc aag ctcttt gcc tct ggg gtg tcc aga gaa cat ggt 2275 Thr Leu Pro Ser Lys Leu PheAla Ser Gly Val Ser Arg Glu His Gly 710 715 720 725 tgc cac agc cac actgac gaa ctg caa gcg ctt gct cct ttg 2317 Cys His Ser His Thr Asp Glu LeuGln Ala Leu Ala Pro Leu 730 735 taaggactcg gaagagtcta agcatcgccactttagctgc tgatctctct ggctccccag 2377 ttcacctctg tggttgtgca gcctacttggagctgaaggc gcacacgggg atatctggaa 2437 tgaaat 2443 12 739 PRT Mouse 12Met Ala Pro Trp Leu Gln Leu Cys Ser Phe Phe Phe Thr Val Asn Ala 1 5 1015 Cys Leu Asn Gly Ser Gln Leu Ala Val Ala Ala Gly Gly Ser Gly Arg 20 2530 Ala Arg Gly Ala Asp Thr Cys Gly Trp Arg Gly Val Gly Pro Ala Ser 35 4045 Arg Asn Ser Gly Leu His Asn Ile Thr Phe Arg Tyr Asp Asn Cys Thr 50 5560 Thr Tyr Leu Asn Pro Gly Gly Gly Lys His Ala Ile Ala Asp Ala Gln 65 7075 80 Asn Ile Thr Ile Ser Gln Tyr Ala Cys His Asp Gln Val Ala Val Thr 8590 95 Ile Leu Trp Ser Pro Gly Ala Leu Gly Ile Glu Phe Leu Lys Gly Phe100 105 110 Arg Val Ile Leu Glu Glu Leu Lys Ser Glu Gly Arg Gln Cys GlnGln 115 120 125 Leu Ile Leu Lys Asp Pro Lys Gln Leu Asn Ser Ser Phe ArgArg Thr 130 135 140 Gly Met Glu Ser Gln Pro Phe Leu Asn Met Lys Phe GluThr Asp Tyr 145 150 155 160 Phe Val Lys Ile Val Pro Phe Pro Ser Ile LysAsn Glu Ser Asn Tyr 165 170 175 His Pro Phe Phe Phe Arg Thr Arg Ala CysAsp Leu Leu Leu Gln Pro 180 185 190 Asp Asn Leu Ala Cys Lys Pro Phe TrpLys Pro Arg Asn Leu Asn Ile 195 200 205 Ser Gln His Gly Ser Asp Met HisVal Ser Phe Asp His Ala Pro Gln 210 215 220 Asn Phe Gly Phe Arg Gly PheHis Val Leu Tyr Lys Leu Lys His Glu 225 230 235 240 Gly Pro Phe Arg ArgArg Thr Cys Arg Gln Asp Gln Asn Thr Glu Thr 245 250 255 Thr Ser Cys LeuLeu Gln Asn Val Ser Pro Gly Asp Tyr Ile Ile Glu 260 265 270 Leu Val AspAsp Ser Asn Thr Thr Arg Lys Ala Ala Gln Tyr Val Val 275 280 285 Lys SerVal Gln Ser Pro Trp Ala Gly Pro Ile Arg Ala Val Ala Ile 290 295 300 ThrVal Pro Leu Val Val Ile Ser Ala Phe Ala Thr Leu Phe Thr Val 305 310 315320 Met Cys Arg Lys Lys Gln Gln Glu Asn Ile Tyr Ser His Leu Asp Glu 325330 335 Glu Ser Pro Glu Ser Ser Thr Tyr Ala Ala Ala Leu Pro Arg Asp Arg340 345 350 Leu Arg Pro Gln Pro Lys Val Phe Leu Cys Tyr Ser Asn Lys AspGly 355 360 365 Gln Asn His Met Asn Val Val Gln Cys Phe Ala Tyr Phe LeuGln Asp 370 375 380 Phe Cys Gly Cys Glu Val Ala Leu Asp Leu Trp Glu AspPhe Ser Leu 385 390 395 400 Cys Arg Glu Gly Gln Arg Glu Trp Ala Ile GlnLys Ile His Glu Ser 405 410 415 Gln Phe Ile Ile Val Val Cys Ser Lys GlyMet Lys Tyr Phe Val Asp 420 425 430 Lys Lys Asn Phe Arg His Lys Gly GlySer Arg Gly Glu Ala Gln Gly 435 440 445 Glu Phe Phe Leu Val Ala Val AlaAla Ile Ala Glu Lys Leu Arg Gln 450 455 460 Ala Lys Gln Ser Ser Ser AlaAla Leu Arg Lys Phe Ile Ala Val Tyr 465 470 475 480 Phe Asp Tyr Ser CysGlu Gly Asp Val Pro Cys Ser Leu Asp Leu Ser 485 490 495 Thr Lys Tyr LysLeu Met Asp His Leu Pro Glu Leu Cys Ala His Leu 500 505 510 His Ser GlyGlu Gln Glu Val Leu Gly Gln His Pro Gly His Ser Ser 515 520 525 Arg ArgAsn Tyr Phe Arg Ser Lys Ser Gly Arg Ser Leu Tyr Val Ala 530 535 540 IleCys Asn Met His Gln Phe Ile Asp Glu Glu Pro Asp Trp Phe Glu 545 550 555560 Lys Gln Phe Ile Pro Phe Gln His Pro Pro Val Arg Tyr Gln Glu Pro 565570 575 Val Leu Glu Lys Phe Asp Ser Gly Leu Val Leu Asn Asp Val Ile Ser580 585 590 Lys Pro Gly Pro Glu Ser Asp Phe Cys Arg Lys Val Glu Ala CysVal 595 600 605 Leu Gly Ala Ala Gly Pro Ala Asp Ser Tyr Ser Tyr Leu GluSer Gln 610 615 620 His Val Gly Leu Asp Gln Asp Thr Glu Ala Gln Pro SerCys Asp Ser 625 630 635 640 Ala Pro Ala Leu Gln Pro Leu Leu His Ala ValLys Ala Gly Ser Pro 645 650 655 Ser Glu Met Pro Arg Asp Ser Gly Ile TyrAsp Ser Ser Val Pro Ser 660 665 670 Ser Glu Leu Ser Leu Pro Leu Met GluGly Leu Ser Pro Asp Gln Ile 675 680 685 Glu Thr Ser Ser Leu Thr Glu SerVal Ser Ser Ser Ser Gly Leu Gly 690 695 700 Glu Glu Asp Pro Pro Thr LeuPro Ser Lys Leu Phe Ala Ser Gly Val 705 710 715 720 Ser Arg Glu His GlyCys His Ser His Thr Asp Glu Leu Gln Ala Leu 725 730 735 Ala Pro Leu 132217 DNA Artificial Sequence This degenerate nucleotide sequence encodesthe amino acid sequence of SEQ ID NO12. 13 atggcnccnt ggytncarytntgywsntty ttyttyacng tnaaygcntg yytnaayggn 60 wsncarytng cngtngcngcnggnggnwsn ggnmgngcnm gnggngcnga yacntgyggn 120 tggmgnggng tnggnccngcnwsnmgnaay wsnggnytnc ayaayathac nttymgntay 180 gayaaytgya cnacntayytnaayccnggn ggnggnaarc aygcnathgc ngaygcncar 240 aayathacna thwsncartaygcntgycay gaycargtng cngtnacnat hytntggwsn 300 ccnggngcny tnggnathgarttyytnaar ggnttymgng tnathytnga rgarytnaar 360 wsngarggnm gncartgycarcarytnath ytnaargayc cnaarcaryt naaywsnwsn 420 ttymgnmgna cnggnatggarwsncarccn ttyytnaaya tgaarttyga racngaytay 480 ttygtnaara thgtnccnttyccnwsnath aaraaygarw snaaytayca yccnttytty 540 ttymgnacnm gngcntgygayytnytnytn carccngaya ayytngcntg yaarccntty 600 tggaarccnm gnaayytnaayathwsncar cayggnwsng ayatgcaygt nwsnttygay 660 caygcnccnc araayttyggnttymgnggn ttycaygtny tntayaaryt naarcaygar 720 ggnccnttym gnmgnmgnacntgymgncar gaycaraaya cngaracnac nwsntgyytn 780 ytncaraayg tnwsnccnggngaytayath athgarytng tngaygayws naayacnacn 840 mgnaargcng cncartaygtngtnaarwsn gtncarwsnc cntgggcngg nccnathmgn 900 gcngtngcna thacngtnccnytngtngtn athwsngcnt tygcnacnyt nttyacngtn 960 atgtgymgna araarcarcargaraayath taywsncayy tngaygarga rwsnccngar 1020 wsnwsnacnt aygcngcngcnytnccnmgn gaymgnytnm gnccncarcc naargtntty 1080 ytntgytayw snaayaargayggncaraay cayatgaayg tngtncartg yttygcntay 1140 ttyytncarg ayttytgyggntgygargtn gcnytngayy tntgggarga yttywsnytn 1200 tgymgngarg gncarmgngartgggcnath caraarathc aygarwsnca rttyathath 1260 gtngtntgyw snaarggnatgaartaytty gtngayaara araayttymg ncayaarggn 1320 ggnwsnmgng gngargcncarggngartty ttyytngtng cngtngcngc nathgcngar 1380 aarytnmgnc argcnaarcarwsnwsnwsn gcngcnytnm gnaarttyat hgcngtntay 1440 ttygaytayw sntgygarggngaygtnccn tgywsnytng ayytnwsnac naartayaar 1500 ytnatggayc ayytnccngarytntgygcn cayytncayw snggngarca rgargtnytn 1560 ggncarcayc cnggncaywsnwsnmgnmgn aaytayttym gnwsnaarws nggnmgnwsn 1620 ytntaygtng cnathtgyaayatgcaycar ttyathgayg argarccnga ytggttygar 1680 aarcarttya thccnttycarcayccnccn gtnmgntayc argarccngt nytngaraar 1740 ttygaywsng gnytngtnytnaaygaygtn athwsnaarc cnggnccnga rwsngaytty 1800 tgymgnaarg tngargcntgygtnytnggn gcngcnggnc cngcngayws ntaywsntay 1860 ytngarwsnc arcaygtnggnytngaycar gayacngarg cncarccnws ntgygaywsn 1920 gcnccngcny tncarccnytnytncaygcn gtnaargcng gnwsnccnws ngaratgccn 1980 mgngaywsng gnathtaygaywsnwsngtn ccnwsnwsng arytnwsnyt nccnytnatg 2040 garggnytnw snccngaycarathgaracn wsnwsnytna cngarwsngt nwsnwsnwsn 2100 wsnggnytng gngargargayccnccnacn ytnccnwsna arytnttygc nwsnggngtn 2160 wsnmgngarc ayggntgycaywsncayacn gaygarytnc argcnytngc nccnytn 2217

We claim:
 1. An isolated nucleic acid molecule encoding a polypeptidewherein the encoded polypeptide comprises amino acid residues 36 to 753of SEQ ID NO:2.
 2. The isolated nucleic acid molecule of claim 1 whereinthe polypeptide comprises SEQ ID NO:2.
 3. The isolated nucleic acidmolecule of claim 1 wherein the polypeptide is SEQ ID NO:2.
 4. Theisolated nucleic acid molecule of claim 1 wherein the isolated nucleicacid molecule comprises nucleotides 191 to 2347 of SEQ ID NO:1.
 5. Theisolated nucleic acid molecule of claim 1 wherein the isolated nucleicacid molecule comprises nucleotides 86 to 2347 of SEQ ID NO:1.
 6. Theisolated nucleic acid molecule of claim 1 wherein the isolated nucleicacid molecule consists of nucleotides 191 to 2347 of SEQ ID NO:1.
 7. Anexpression vector comprising the following operably linked elements: atranscription promoter; a DNA segment encoding a polypeptide wherein theencoded polypeptide comprises amino acid residues 36 to 753 of SEQ IDNO:2; and a transcription terminator.
 8. The expression vector of claim7 wherein the DNA segment further encodes a secretory signal sequenceoperably linked to the polypeptide.
 9. The expression vector of claim 8wherein the secretory signal sequence comprises amino acid residues 1 to35 of SEQ ID NO:2.
 10. A recombinant host cell comprising the expressionvector of claim
 7. 11. A method of using the expression vector of claim7 to produce a polypeptide that comprises amino acid residues 36 to 753of SEQ ID NO:2, comprising culturing recombinant host cells thatcomprise the expression vector and that produce the polypeptide.
 12. Themethod of claim 11 further comprising isolating the polypeptide from thecultured recombinant host cells.
 13. An isolated polynucleotide encodinga fusion protein wherein the encoded fusion protein comprises a firstportion and a second portion joined by a peptide bond, wherein the firstportion comprises amino acid residues 36 to 753 of SEQ ID NO:2, andwherein the second portion comprises another polypeptide.
 14. Theisolated polynucleotide of claim 13 wherein the polynucleotide furtherencodes a secretory signal sequence consisting of amino acid residues 1to 25 of SEQ ID NO:2, and wherein the secretory signal sequence isoperably linked to the first portion and the second portion.